Destabilizing NF1 variants act in a dominant negative manner through neurofibromin dimerization.

The majority of pathogenic mutations in the neurofibromatosis type I (NF1) gene reduce total neurofibromin protein expression through premature truncation or microdeletion, but it is less well understood how loss-of-function missense variants drive NF1 disease. We have found that patient variants in codons 844 to 848, which correlate with a severe phenotype, cause protein instability and exert an additional dominant-negative action whereby wild-type neurofibromin also becomes destabilized through protein dimerization. We have used our neurofibromin cryogenic electron microscopy structure to predict and validate other patient variants that act through a similar mechanism. This provides a foundation for understanding genotype-phenotype correlations and has important implications for patient counseling, disease management, and therapeutics.

Classical RAS proteins are not essential for paradoxical ERK activation induced by RAF inhibitors.

RAF inhibitors unexpectedly induce ERK signaling in normal and tumor cells with elevated RAS activity. Paradoxical activation is believed to be RAS dependent. In this study, we showed that LY3009120, a pan-RAF inhibitor, can unexpectedly cause paradoxical ERK activation in KRASG12C-dependent lung cancer cell lines, when KRAS is inhibited by ARS1620, a KRASG12C inhibitor. Using H/N/KRAS-less mouse embryonic fibroblasts, we discovered that classical RAS proteins are not essential for RAF inhibitor-induced paradoxical ERK signaling. In their absence, RAF inhibitors can induce ERK phosphorylation, ERK target gene transcription, and cell proliferation. We further showed that the MRAS/SHOC2 complex is required for this process. This study highlights the complexity of the allosteric RAF regulation by RAF inhibitors, and the importance of other RAS-related proteins in this process.

Unexpected tobacco etch virus (TEV) protease cleavage of recombinant human proteins.

The tobacco etch virus (TEV) protease is a commonly used reagent for removal of solubility and purification tags from recombinant proteins and is cited as being highly specific for its canonical cleavage site. Flexibility in some amino acids within this recognition sequence has been described in the literature but researchers generally assume few native human proteins will carry off-target sequences for TEV cleavage. We report here the aberrant cleavage of three human proteins with non-canonical TEV protease cleavage sites and identify broader sequence specificity rules that can be used to predict unwanted cleavage of recombinant proteins. Using these rules, 456 human proteins were identified that could be substrates for unwanted TEV protease cleavage.

Adapting recombinant bacterial alkaline phosphatase for nucleotide exchange of small GTPases.

The small GTPase Rat sarcoma virus proteins (RAS) are key regulators of cell growth and involved in 20-30% of cancers. RAS switches between its active state and inactive state via exchange of GTP (active) and GDP (inactive). Therefore, to study active protein, it needs to undergo nucleotide exchange to a non-hydrolysable GTP analog. Calf intestine alkaline phosphatase bound to agarose beads (CIP-agarose) is regularly used in a nucleotide exchange protocol to replace GDP with a non-hydrolysable analog. Due to pandemic supply problems and product shortages, we found the need for an alternative to this commercially available product. Here we describe how we generated a bacterial alkaline phosphatase (BAP) with an affinity tag bound to an agarose bead. This BAP completely exchanges the nucleotide in our samples, thereby demonstrating an alternative to the commercially available product using generally available laboratory equipment.

Polycistronic baculovirus expression of SUGT1 enables high-yield production of recombinant leucine-rich repeat proteins and protein complexes.

The SHOC2-MRAS-PPP1CA (SMP) complex is a holoenzyme that plays a vital role in the MAP kinase signaling pathway. Previous attempts to produce this challenging three-protein complex have relied on co-infection with multiple viruses and the use of affinity tags to attempt to isolate functional recombinant protein complexes. Leucine-rich repeat containing proteins have been historically challenging to express, and we hypothesized that co-expression of appropriate chaperones may be necessary for optimal production. We describe here how the SUGT1 chaperone can, in conjunction with polycistronic protein expression in baculovirus-infected insect cells, dramatically enhance production yield and quality of recombinant SHOC2, the SMP complex, and other leucine-rich repeat proteins.

Refining the N-Termini of the SARS-CoV-2 Spike Protein and Its Discrete Receptor-Binding Domain.

Previous work employing five SARS-CoV-2 spike protein receptor-binding domain (RBD) constructs, comprising versions originally developed by Mt. Sinai or the Ragon Institute and later optimized in-house, revealed potential heterogeneity which led to questions regarding variable seropositivity assay performance. Each construct was subjected to N-deglycosylation and subsequent intact mass analysis, revealing significant deviations from predicted theoretical mass for all five proteins. Complementary tandem MS/MS analysis revealed the presence of an additional pyroGlu residue on the N-termini of the two Mt. Sinai RBD constructs, as well as on the N-terminus of the full-length spike protein from which they were derived, thus explaining the observed mass shift and definitively establishing the spike protein N-terminal sequence. Moreover, the observed mass additions for the three Ragon Institute RBD constructs were identified as variable N-terminal cleavage points within the signal peptide sequence employed for recombinant expression. To resolve this issue and minimize heterogeneity for further seropositivity assay development, the best-performing RBD construct was further optimized to exhibit complete homogeneity, as determined by both intact mass and tandem MS/MS analysis. This new RBD construct has been validated for seropositivity assay performance, is available to the greater scientific community, and is recommended for use in future assay development.

Improved production of SARS-CoV-2 spike receptor-binding domain (RBD) for serology assays.

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a commonly used antigen for serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Different versions of the RBD protein have been developed and utilized in assays, with higher sensitivity attributed to particular forms of the protein. To improve the yield of these high-sensitivity forms of RBD and support the increased demand for this antigen in serology assays, we investigated several protein expression variables including DNA elements such as promoters and signal peptides, cell culture expression parameters, and purification processes. Through this investigation, we developed a simplified and robust purification strategy that consistently resulted in high levels of the high-sensitivity form of RBD and demonstrated that a carboxyterminal tag is responsible for the increased sensitivity in the ELISA. These improved reagents and processes produce high-quality proteins which are functional in serology assays and can be used to investigate seropositivity to SARS-CoV-2 infection.

Optimizing high-yield production of SARS-CoV-2 soluble spike trimers for serology assays.

The SARS-CoV-2 spike trimer is the primary antigen for several serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Until stable cell lines are developed to increase the titer of this secreted protein in mammalian cell culture, the low yield of spike protein produced from transient transfection of HEK293 cells will be a limiting factor for these assays. To improve the yield of spike protein and support the high demand for antigens in serology assays, we investigated several recombinant protein expression variables by altering the incubation temperature, harvest time, chromatography strategy, and final protein manipulation. Through this investigation, we developed a simplified and robust purification strategy that consistently yields 5 mg of protein per liter of expression culture for two commonly used forms of the SARS-CoV-2 spike protein. We show that these proteins form well-behaved stable trimers and are consistently functional in serology assays across multiple protein production lots.

Exercise opens a temporal window for enhanced cognitive improvement from subsequent physical activity.

The beneficial effects of exercise on cognition are well established; however specific exercise parameters regarding the frequency and duration of physical activity that provide optimal cognitive health have not been well defined. Here, we explore the effects of the duration of exercise and sedentary periods on long-term object location memory (OLM) in mice. We use a weak object location training paradigm that is subthreshold for long-term memory formation in sedentary controls, and demonstrate that exercise enables long-term memories to form. We show that 14- and 21-d of running wheel access enables mice to discriminate between familiar and novel object locations after a 24 h delay, while 2- or 7-d running wheel access provides insufficient exercise for such memory enhancement using the subthreshold learning paradigm. After 14- and 21-d of wheel running, exercise-induced cognitive enhancement then decays back to baseline performance following 3-d of sedentary activity. However, exercise-induced cognitive enhancement can be reactivated by an additional period of just 2 d exercise, previously shown to be insufficient to induce cognitive enhancement on its own. The reactivating period of exercise is capable of enhancing memory after three- or seven-sedentary days, but not 14-d. These data suggest a type of "molecular memory" for the exercise stimulus, in that once exercise duration reaches a certain threshold, it establishes a temporal window during which subsequent low-level exercise can capitalize on the neurobiological adaptations induced by the initial period of exercise, enabling it to maintain the benefits on cognitive function. These findings provide new information that may help to guide future clinical studies in exercise.

Creation of an Aesthetic Male Nipple Areolar Complex in Female-to-Male Transgender Chest Reconstruction.

BACKGROUND: Female-to-male chest wall reconstruction is becoming more common, but while there is a growing body of the literature describing technique and algorithms, little detail is written on methods for creating a male appearing nipple areolar complex (NAC) from a female NAC utilizing free nipple graft techniques. Incorrect positioning of the NAC on the chest wall and suboptimal shaping and sizing of the NAC are common pitfalls in male NAC creation. METHODS: With this paper, we present techniques for nipple grafting to achieve improved male appearing NACs, as well as a simple, reproducible method for appropriate placement of the NAC relative to the borders of the pectoralis muscle. To validate our technique, we performed photographic analysis of 64 NACs in 32 volunteers with BMI of 25 or less. RESULTS: The anatomic study determined the cis-male nipple to be positioned on average 2.5 cm medial to the lateral border of the pectoralis muscle and 2.4 cm above the inferior pectoralis insertion. This supports our surgical technique of positioning the NAC in relation to the pectoralis borders rather than previously advocated anatomic landmarks. We also present reliable techniques for creating a round or horizontally oval final NAC shape as well as a composite grafting technique for cases of large papillae. CONCLUSIONS: Our anatomic study supports placement of the male NAC relative to lateral and inferior borders of the pectoralis muscle. Nipple grafting techniques presented allow for a simple and reproducible method of creating an aesthetic male NAC shape in female-to-male transgender chest reconstruction. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

FLAG-KRAS4B as a Model System for KRAS4B Proteoform and PTM Evaluation by Mass Spectrometry.

Prior analysis of intact and modified protein forms (proteoforms) of KRAS4B isolated from cell lines and tumor samples by top-down mass spectrometry revealed the presence of novel posttranslational modifications (PTMs) and potential evidence of context-specific KRAS4B modifications. However, low endogenous proteoform signal resulted in ineffective characterization, making it difficult to visualize less abundant PTMs or perform follow-up PTM validation using standard proteomic workflows. The NCI RAS Initiative has developed a model system, whereby KRAS4B bearing an N-terminal FLAG tag can be stably expressed within a panel of cancer cell lines. Herein, we present a method for combining immunoprecipitation with complementary proteomic methods to directly analyze N-terminally FLAG-tagged KRAS4B proteoforms and PTMs. We provide detailed protocols for FLAG-KRAS4B purification, proteoform analysis by targeted top-down LC-MS/MS, and validation of abundant PTMs by bottom-up LC-MS/MS with example results.

Leveraging mHealth and Wearable Sensors to Manage Alcohol Use Disorders: A Systematic Literature Review.

BACKGROUND: Alcohol use disorder (AUD) is a condition prevalent in many countries around the world, and the public burden of its treatment is close to $130 billion. mHealth offers several possible interventions to assist in the treatment of AUD. OBJECTIVES: To analyze the effectiveness of mHealth and wearable sensors to manage AUD from evidence published over the last 10 years. METHODS: Following the Kruse Protocol and PRISMA 2020, four databases were queried (PubMed, CINAHL, Web of Science, and Science Direct) to identify studies with strong methodologies (n = 25). RESULTS: Five interventions were identified, and 20/25 were effective at reducing alcohol consumption. Other interventions reported a decrease in depression and an increase in medication compliance. Primary barriers to the adoption of mHealth interventions are a requirement to train users, some are equally as effective as the traditional means of treatment, cost, and computer literacy. CONCLUSION: While not all mHealth interventions demonstrated statistically significant reduction in alcohol consumption, most are still clinically effective to treat AUD and provide a patient with their preference of a technologically inclined treatment Most interventions require training of users and some technology literacy, the barriers identified were very few compared with the litany of positive results.

Anterior versus posterior entry site for ventriculoperitoneal shunt insertion: a randomized controlled trial by the Hydrocephalus Clinical Research Network.

OBJECTIVE: The primary objective of this trial was to determine if shunt entry site affects the risk of shunt failure. METHODS: The authors performed a parallel-design randomized controlled trial with an equal allocation of patients who received shunt placement via the anterior entry site and patients who received shunt placement via the posterior entry site. All patients were children with symptoms or signs of hydrocephalus and ventriculomegaly. Patients were ineligible if they had a prior history of shunt insertion. Patients received a ventriculoperitoneal shunt after randomization; randomization was stratified by surgeon. The primary outcome was shunt failure. The planned minimum follow-up was 18 months. The trial was designed to achieve high power to detect a 10% or greater absolute difference in the shunt failure rate at 1 year. An independent, blinded adjudication committee determined eligibility and the primary outcome. The study was conducted by the Hydrocephalus Clinical Research Network. RESULTS: The study randomized 467 pediatric patients at 14 tertiary care pediatric hospitals in North America from April 2015 to January 2019. The adjudication committee, blinded to intervention, excluded 7 patients in each group for not meeting the study inclusion criteria. For the primary analysis, there were 229 patients in the posterior group and 224 patients in the anterior group. The median patient age was 1.3 months, and the most common etiologies of hydrocephalus were postintraventricular hemorrhage secondary to prematurity (32.7%), myelomeningocele (16.8%), and aqueductal stenosis (10.8%). There was no significant difference in the time to shunt failure between the entry sites (log-rank test, stratified by age < 6 months and ≥ 6 months; p = 0.061). The hazard ratio (HR) of a posterior shunt relative to an anterior shunt was calculated using a univariable Cox regression model and was nonsignificant (HR 1.35, 95% CI, 0.98-1.85; p = 0.062). No significant difference was found between entry sites for the surgery duration, number of ventricular catheter passes, ventricular catheter location, and hospital length of stay. There were no significant differences between entry sites for intraoperative complications, postoperative CSF leaks, pseudomeningoceles, shunt infections, skull fractures, postoperative seizures, new-onset epilepsy, or intracranial hemorrhages. CONCLUSIONS: This randomized controlled trial comparing the anterior and posterior shunt entry sites has demonstrated no significant difference in the time to shunt failure. Anterior and posterior entry site surgeries were found to have similar outcomes and similar complication rates.

Social-emotional functioning in pediatric hydrocephalus: comparison of the Hydrocephalus Outcome Questionnaire to the Behavior Assessment System for Children.

OBJECTIVE: Hydrocephalus can impact all areas of health, including physical, cognitive, and social-emotional functioning. The social-emotional health of children who have had surgery for hydrocephalus is not well characterized. In this study, the authors sought to examine social-emotional functioning using the Behavior Assessment System for Children, Third Edition (BASC-3) and the Hydrocephalus Outcome Questionnaire (HOQ) in 66 children aged 5 to 17 years. METHODS: Caregivers of pediatric patients with hydrocephalus completed the BASC-3 and the HOQ. BASC-3 internalizing, externalizing, and executive functioning caregiver-reported scores were compared with the BASC-3 normative sample using one-sample t-tests to evaluate overall social-emotional functioning. BASC-3 scores were correlated with the social-emotional domain of the HOQ using Pearson's r to determine if the HOQ accurately captured the social-emotional functioning of children with hydrocephalus in a neurosurgery setting. BASC-3 and HOQ scores of children with different etiologies of hydrocephalus were compared using the Kruskal-Wallis one-way analysis of variance to determine if differences existed between the following etiologies: intraventricular hemorrhage secondary to prematurity, myelomeningocele, communicating congenital hydrocephalus, aqueductal stenosis, or other. RESULTS: Children with hydrocephalus of all etiologies had more difficulties with social-emotional functioning compared with normative populations. Children with different hydrocephalus etiologies differed in executive functioning and overall HOQ scores but not in internalizing symptoms, externalizing symptoms, or social-emotional HOQ scores. The social-emotional domain of the HOQ correlated more strongly with the BASC-3 than did the physical and cognitive domains. CONCLUSIONS: These results have provided evidence that children who have had surgery for hydrocephalus may be at increased risk of social-emotional and behavioral difficulties, but etiology may not be particularly helpful in predicting what kinds or degree of difficulty. The results of this study also support the convergent and divergent validity of the social-emotional domain of the HOQ.

Standardization of ELISA protocols for serosurveys of the SARS-CoV-2 pandemic using clinical and at-home blood sampling.

The extent of SARS-CoV-2 infection throughout the United States population is currently unknown. High quality serology is key to avoiding medically costly diagnostic errors, as well as to assuring properly informed public health decisions. Here, we present an optimized ELISA-based serology protocol, from antigen production to data analyses, that helps define thresholds for IgG and IgM seropositivity with high specificities. Validation of this protocol is performed using traditionally collected serum as well as dried blood on mail-in blood sampling kits. Archival (pre-2019) samples are used as negative controls, and convalescent, PCR-diagnosed COVID-19 patient samples serve as positive controls. Using this protocol, minimal cross-reactivity is observed for the spike proteins of MERS, SARS1, OC43 and HKU1 viruses, and no cross reactivity is observed with anti-influenza A H1N1 HAI. Our protocol may thus help provide standardized, population-based data on the extent of SARS-CoV-2 seropositivity, immunity and infection.

Mapping a Pandemic: SARS-CoV-2 Seropositivity in the United States.

Asymptomatic SARS-CoV-2 infection and delayed implementation of diagnostics have led to poorly defined viral prevalence rates. To address this, we analyzed seropositivity in US adults who have not previously been diagnosed with COVID-19. Individuals with characteristics that reflect the US population (n = 11,382) and who had not previously been diagnosed with COVID-19 were selected by quota sampling from 241,424 volunteers (ClinicalTrials.gov NCT04334954). Enrolled participants provided medical, geographic, demographic, and socioeconomic information and 9,028 blood samples. The majority (88.7%) of samples were collected between May 10th and July 31st, 2020. Samples were analyzed via ELISA for anti-Spike and anti-RBD antibodies. Estimation of seroprevalence was performed by using a weighted analysis to reflect the US population. We detected an undiagnosed seropositivity rate of 4.6% (95% CI: 2.6 - 6.5%). There was distinct regional variability, with heightened seropositivity in locations of early outbreaks. Subgroup analysis demonstrated that the highest estimated undiagnosed seropositivity within groups was detected in younger participants (ages 18-45, 5.9%), females (5.5%), Black/African American (14.2%), Hispanic (6.1%), and Urban residents (5.3%), and lower undiagnosed seropositivity in those with chronic diseases. During the first wave of infection over the spring/summer of 2020 an estimate of 4.6% of adults had a prior undiagnosed SARS-CoV-2 infection. These data indicate that there were 4.8 (95% CI: 2.8-6.8) undiagnosed cases for every diagnosed case of COVID-19 during this same time period in the United States, and an estimated 16.8 million undiagnosed cases by mid-July 2020.

Undiagnosed SARS-CoV-2 seropositivity during the first 6 months of the COVID-19 pandemic in the United States.

Asymptomatic SARS-CoV-2 infection and delayed implementation of diagnostics have led to poorly defined viral prevalence rates in the United States and elsewhere. To address this, we analyzed seropositivity in 9089 adults in the United States who had not been diagnosed previously with COVID-19. Individuals with characteristics that reflected the U.S. population (n = 27,716) were selected by quota sampling from 462,949 volunteers. Enrolled participants (n = 11,382) provided medical, geographic, demographic, and socioeconomic information and dried blood samples. Survey questions coincident with the Behavioral Risk Factor Surveillance System survey, a large probability-based national survey, were used to adjust for selection bias. Most blood samples (88.7%) were collected between 10 May and 31 July 2020 and were processed using ELISA to measure seropositivity (IgG and IgM antibodies against SARS-CoV-2 spike protein and the spike protein receptor binding domain). The overall weighted undiagnosed seropositivity estimate was 4.6% (95% CI, 2.6 to 6.5%), with race, age, sex, ethnicity, and urban/rural subgroup estimates ranging from 1.1% to 14.2%. The highest seropositivity estimates were in African American participants; younger, female, and Hispanic participants; and residents of urban centers. These data indicate that there were 4.8 undiagnosed SARS-CoV-2 infections for every diagnosed case of COVID-19, and an estimated 16.8 million infections were undiagnosed by mid-July 2020 in the United States.

Improved production of SARS-CoV-2 spike receptor-binding domain (RBD) for serology assays.

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a commonly used antigen for serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Different versions of the RBD protein have been developed and utilized in assays, with higher sensitivity attributed to particular forms of the protein. To improve the yield of these high-sensitivity forms of RBD and support the increased demand for this antigen in serology assays, we investigated several protein expression variables including DNA elements such as promoters and signal peptides, cell culture expression parameters, and purification processes. Through this investigation, we developed a simplified and robust purification strategy that consistently resulted in high levels of the high-sensitivity form of RBD and demonstrated that a carboxyterminal tag is responsible for the increased sensitivity in the ELISA. These improved reagents and processes produce high-quality proteins which are functional in serology assays and can be used to investigate seropositivity to SARS-CoV-2 infection. Highlights: Improved yields of SARS-CoV-2 spike RBD through modification of DNA constructs and purification parametersTwo versions of RBD show different sensitivity in serology assaysYields of greater than 50 mg/l obtained under optimal conditionsMagnetic bead purification technology improves throughput of protein production.

Optimizing high-yield production of SARS-CoV-2 soluble spike trimers for serology assays.

The SARS-CoV-2 spike trimer is the primary antigen for several serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Until stable cell lines are developed to increase the titer of this secreted protein in mammalian cell culture, the low yield of spike protein produced from transient transfection of HEK293 cells will be a limiting factor for these assays. To improve the yield of spike protein and support the high demand for antigens in serology assays, we investigated several recombinant protein expression variables by altering the incubation temperature, harvest time, chromatography strategy, and final protein manipulation. Through this investigation, we developed a simplified and robust purification strategy that consistently yields 5 mg of protein per liter of expression culture for two commonly used forms of the SARS-CoV-2 spike protein. We show that these proteins form well-behaved stable trimers and are consistently functional in serology assays across multiple protein production lots.

Membrane interactions of the globular domain and the hypervariable region of KRAS4b define its unique diffusion behavior.

The RAS proteins are GTP-dependent switches that regulate signaling pathways and are frequently mutated in cancer. RAS proteins concentrate in the plasma membrane via lipid-tethers and hypervariable region side-chain interactions in distinct nano-domains. However, little is known about RAS membrane dynamics and the details of RAS activation of downstream signaling. Here, we characterize RAS in live human and mouse cells using single-molecule-tracking methods and estimate RAS mobility parameters. KRAS4b exhibits confined mobility with three diffusive states distinct from the other RAS isoforms (KRAS4a, NRAS, and HRAS); and although most of the amino acid differences between RAS isoforms lie within the hypervariable region, the additional confinement of KRAS4b is largely determined by the protein's globular domain. To understand the altered mobility of an oncogenic KRAS4b, we used complementary experimental and molecular dynamics simulation approaches to reveal a detailed mechanism.