Structural Insights into Dopamine Receptor-Ligand Interactions: From Agonists to Antagonists.

This study explores the intricacies of dopamine receptor-ligand interactions, focusing on the D1R and D5R subtypes. Using molecular modeling techniques, we investigate the binding of the pan-agonist rotigotine, revealing a universal binding mode at the orthosteric binding pocket (OBP). Additionally, we analyze the stability of antagonist-receptor complexes with SKF83566 and SCH23390. By examining the impact of specific mutations on ligand-receptor interactions through computational simulations and thermostability assays, we gain insights into binding stability. Our research also delves into the structural and energetic aspects of antagonist binding to D1R and D5R in their inactive states. These findings enhance our understanding of dopamine receptor pharmacology and hold promise for drug development in central nervous system disorders, opening doors to future research and innovation in this field.

Structure-Based Lead Optimization of Enterovirus D68 2A Protease Inhibitors.

Enterovirus D68 (EV-D68) virus is a nonpolio enterovirus that typically causes respiratory illness and, in severe cases, can lead to paralysis and death in children. There is currently no vaccine or antiviral for EV-D68. We previously discovered the viral 2A protease (2Apro) as a viable antiviral drug target and identified telaprevir as a 2Apro inhibitor. 2Apro is a viral cysteine protease that cleaves the viral VP1-2A polyprotein junction. In this study, we report the X-ray crystal structures of EV-D68 2Apro, wild-type, and the C107A mutant and the structure-based lead optimization of telaprevir. Guided by the X-ray crystal structure, we predicted the binding pose of telaprevir in 2Apro using molecular dynamics simulations. We then utilized this model to inform structure-based optimization of the telaprevir's reactive warhead and P1-P4 substitutions. These efforts led to the discovery of 2Apro inhibitors with improved antiviral activity than telaprevir. These compounds represent promising lead compounds for further development as EV-D68 antivirals.

CROSS-CULTURAL ADAPTATION OF THE HOSPITAL ETHICAL CLIMATE SURVEY TO BRAZIL.

Background and Purpose: Adapting cross-culturally the Hospital Ethical Climate Survey to Brazilian nurses. Methods: A methodological study comprising translation, back translation, multidisciplinary committee, expert panel, pilot test and validation of the instrument. The validation was carried out with 269 nurses of a university hospital in the South of Brazil. Results: In the validation step, the quadratic weighted Kappa test-retest and the correlation coefficient ranged from 0.15 to 0.74. All factor loadings were higher than 0.4, ranging from 0.445 to 0.859. The Portuguese version of the instrument had a 0.93 Cronbach's alpha and the confirmatory analysis demonstrated the model's suitability with five factors and 26 items validated. Conclusions: The version of the instrument adapted to Brazilian Portuguese was considered valid and reliable in this sample.

Let's Talk About It: Addressing Microaggressions in Occupational Therapy Education.

There is an increasing need to diversify the profession of occupational therapy. To do this, we need to examine the pathway to becoming an occupational therapy practitioner in our education programs. Programs must go beyond a focus on diversity and place an equal emphasis on equity and inclusion. Underrepresented minority students report increased racial bias and a high occurrence of microaggressions in higher education programs, including occupational therapy. These microaggressions are often not addressed and can lead to increased stress, insecurity, and marginalization, as well as decreased retention rates. It is imperative that faculty examine the equity and inclusivity of their programs, and they should be supported in addressing their own implicit bias. Faculty, students, and staff should be equipped to address microaggressions as they occur. This article identifies examples of microaggressions and suggests strategies to address implicit bias and microaggressions in occupational therapy programs.

Promoting Safe Mobility Strategies for partnering with caregivers to maximize older adults' functional ability.

This article is the fifth in a series, Supporting Family Caregivers in the 4Ms of an Age-Friendly Health System, published in collaboration with the AARP Public Policy Institute as part of the ongoing Supporting Family Caregivers: No Longer Home Alone series. The 4Ms of an Age-Friendly Health System (What Matters, Medication, Mentation, and Mobility) is an evidence-based framework for assessing and acting on critical issues in the care of older adults across settings and transitions of care. Engaging the health care team, including older adults and their family caregivers, with the 4Ms framework can help to ensure that every older adult gets the best care possible, is not harmed by health care, and is satisfied with the care they receive. The articles in this series present considerations for implementing the 4Ms framework in the inpatient hospital setting and incorporating family caregivers in doing so. Resources for both nurses and family caregivers, including a series of accompanying videos developed by AARP and the Rush Center for Excellence in Aging and funded by The John A. Hartford Foundation, are also provided. Nurses should read the articles first, so they understand how best to help family caregivers. Then they can refer caregivers to the informational tear sheet-Information for Family Caregivers-and instructional videos, encouraging them to ask questions. For additional information, see Resources for Nurses. Cite this article as: Olson,L.M., et al. Promote Safe Mobility. Am J Nurs 2022; 122(7): 46-52.

Hospital ethical climate survey - selected psychometric properties of the scale and results among polish nurses and midwives.

BACKGROUND: The hospital ethical climate affects the quality of nursing care. A positive ethical climate is likely to reduce the proportion of those who consider leaving the profession, so it is necessary to develop tools which will enable assessment and analysis of the hospital ethical climate. The aim of this study was to examine selected psychometric properties of the Polish version of the Hospital Ethical Climate Survey, assess the hospital ethical climate perceived by nurses and midwives from Polish hospitals, and to determine its correlations with job-related variables. METHODS: A cross-sectional study among 558 nurses and midwives working in hospitals in Poland. RESULTS: The 21-item model showed acceptable model fitness between the hypothetical model of ethical climate and the data in the study. Five items with low factor loadings were removed from the study. The internal consistency was satisfactory (0.93). The mean score for the overall hospital ethical climate was 3.62. The highest mean score of hospital ethical climate in the present study was found in the "peers" subscale and the lowest in the "physicians" subscale. A positive correlation was found between overall hospital ethical climate and respondents' satisfaction with work, salary, and working time. The hospital ethical climate was associated with problems found in nurses and midwives' work, such as: limited time for direct face-to-face care, the lack of equipment and resources to provide high-quality health care, strained relations with hospital managers and other health care professionals, limitations to one's own competences or those of other medical professionals, moral dilemmas related to patient care, the low prestige of nurses'/midwives' work, physical and mental burden, and the risk of making a mistake. CONCLUSION: The Polish 21-item version of the Hospital Ethical Climate Survey is a reliable tool. Correlations revealed that relationships with managers and physicians, and working conditions should be improved in order for the hospital ethical climate to improve.

Promoting Safe Mobility.

This article is the fifth in a series, Supporting Family Caregivers in the 4Ms of an Age-Friendly Health System, published in collaboration with the AARP Public Policy Institute as part of the ongoing Supporting Family Caregivers: No Longer Home Alone series. The 4Ms of an Age-Friendly Health System (What Matters, Medication, Mentation, and Mobility) is an evidence-based framework for assessing and acting on critical issues in the care of older adults across settings and transitions of care. Engaging the health care team, including older adults and their family caregivers, with the 4Ms framework can help to ensure that every older adult gets the best care possible, is not harmed by health care, and is satisfied with the care they receive. The articles in this series present considerations for implementing the 4Ms framework in the inpatient hospital setting and incorporating family caregivers in doing so. Resources for both nurses and family caregivers, including a series of accompanying videos developed by AARP and the Rush Center for Excellence in Aging and funded by The John A. Hartford Foundation, are also provided. Nurses should read the articles first, so they understand how best to help family caregivers. Then they can refer caregivers to the informational tear sheet-Information for Family Caregivers-and instructional videos, encouraging them to ask questions. For additional information, see Resources for Nurses.

Evaluation of Incise Drape Lift Using 2% Chlorhexidine Gluconate/70% Isopropyl Alcohol Preoperative Skin Preparations in a Human Volunteer Knee Model.

Background: Before surgery, skin is prepped with antiseptics to reduce risk of surgical site infections. An incise drape can be used as an additional modality to immobilize any remaining bacteria. Good adhesion of this drape is critical for infection prevention. Methods: This is a randomized controlled study using a human volunteer knee model (n = 30) to evaluate the adhesion performance of an incise drape comparing 2 skin preparations. A new investigational 2% chlorhexidine gluconate/70% isopropyl alcohol skin prep (prep A) was compared with an existing skin prep containing the same active agents (prep B). Two samples of an iodine-impregnated incise drape were placed on each knee after prepping. Knees were flexed in dry conditions, under a saline-soaked gauze, and after saline lavage. The frequency of drape lift was recorded after each challenge. Results: After dry flex, 4 of 60 samples (6.7%) had lifted on prep A and 0 on prep B (P = .125). After wet flex, 20 of 60 samples (33%) had lifted on prep A, whereas 42 of 60 samples (70%) had lifted on prep B (P < .0001). After lavage, 23 of 60 samples (38%) had lifted on prep A, whereas 48 of 60 samples (80%) had lifted on prep B (P < .0001). Both preps were well tolerated with minimal erythema and no edema, rash, dryness, or denudation observed. No adverse events were reported. Conclusions: Prep A resulted in reduced frequency of incise drape lift from skin under wet conditions in this model compared with prep B.

The Lived Experience of Sexuality Among Adults With Intellectual and Developmental Disabilities: A Scoping Review.

IMPORTANCE: Sexuality is an important part of the human experience. However, little is known about the experience of sexuality from the perspective of adults with intellectual and developmental disabilities (IDD). OBJECTIVE: To review evidence about the lived experience of sexuality among adults with IDD to inform future research and practice. DATA SOURCES: We located articles using search terms summarized into three categories: intellectual disability, sexual activity, and feelings/opinions. Articles were indexed in the following electronic databases: CINAHL, Scopus, PubMed, and PsycINFO. Study Selection and Data Collection: Arksey and O'Malley's methodology was used to review scientific literature published between 2008 and 2018. The studies were in English, were located in peer-reviewed journals, and described the experience of sexuality from the perspective of people with IDD or observations of expressed sexuality. FINDINGS: Seventeen articles met inclusion criteria. Four main themes emerged: intimate relationships, oppressed sexual activity, knowledge, and identity. Findings indicate that sexuality is a part of the lives of many adults with IDD, regardless of their engagement in sexual activity. CONCLUSIONS AND RELEVANCE: Barriers to expression of sexuality exist for people with IDD. Some of these barriers are internal, although many are external. The results provide support for addressing sexuality among adults with IDD. What This Article Adds: This review provides evidence to support occupational therapy practitioners in including sexuality as a part of their holistic view of clients and as an area for advocacy.

Tissue plasminogen activator is a ligand of cation-independent mannose 6-phosphate receptor and consists of glycoforms that contain mannose 6-phosphate.

Plasmin is the key enzyme in fibrinolysis. Upon interaction with plasminogen activators, the zymogen plasminogen is converted to active plasmin. Some studies indicate plasminogen activation is regulated by cation-independent mannose 6-phosphate receptor (CI-MPR), a protein that facilitates lysosomal enzyme trafficking and insulin-like growth factor 2 downregulation. Plasminogen regulation may be accomplished by CI-MPR binding to plasminogen or urokinase plasminogen activator receptor. We asked whether other members of the plasminogen activation system, such as tissue plasminogen activator (tPA), also interact with CI-MPR. Because tPA is a glycoprotein with three N-linked glycosylation sites, we hypothesized that tPA contains mannose 6-phosphate (M6P) and binds CI-MPR in a M6P-dependent manner. Using surface plasmon resonance, we found that two sources of tPA bound the extracellular region of human and bovine CI-MPR with low-mid nanomolar affinities. Binding was partially inhibited with phosphatase treatment or M6P. Subsequent studies revealed that the five N-terminal domains of CI-MPR were sufficient for tPA binding, and this interaction was also partially mediated by M6P. The three glycosylation sites of tPA were analyzed by mass spectrometry, and glycoforms containing M6P and M6P-N-acetylglucosamine were identified at position N448 of tPA. In summary, we found that tPA contains M6P and is a CI-MPR ligand.

Allosteric regulation of lysosomal enzyme recognition by the cation-independent mannose 6-phosphate receptor.

The cation-independent mannose 6-phosphate receptor (CI-MPR, IGF2 receptor or CD222), is a multifunctional glycoprotein required for normal development. Through the receptor's ability to bind unrelated extracellular and intracellular ligands, it participates in numerous functions including protein trafficking, lysosomal biogenesis, and regulation of cell growth. Clinically, endogenous CI-MPR delivers infused recombinant enzymes to lysosomes in the treatment of lysosomal storage diseases. Although four of the 15 domains comprising CI-MPR's extracellular region bind phosphorylated glycans on lysosomal enzymes, knowledge of how CI-MPR interacts with ~60 different lysosomal enzymes is limited. Here, we show by electron microscopy and hydroxyl radical protein footprinting that the N-terminal region of CI-MPR undergoes dynamic conformational changes as a consequence of ligand binding and different pH conditions. These data, coupled with X-ray crystallography, surface plasmon resonance and molecular modeling, allow us to propose a model explaining how high-affinity carbohydrate binding is achieved through allosteric domain cooperativity.

Inhibitors of tri- and tetra- polyamine oxidation, but not diamine oxidation, impair the initial stages of wound-induced suberization.

The mechanisms regulating, and modulating potato wound-healing processes are of great importance in reducing tuber infections, reducing shrinkage and maintaining quality and nutritional value for growers and consumers. Wound-induced changes in tuber polyamine metabolism have been linked to the modulation of wound healing (WH) and in possibly providing the crucial amount of H2O2 required for suberization processes. In this investigation we determined the effect of inhibition of specific steps within the pathway of polyamine metabolism on polyamine content and the initial accumulation of suberin polyphenolics (SPP) during WH. The accumulation of SPP represents a critical part of the beginning or inchoate phase of tuber WH during closing-layer formation because it serves as a barrier to bacterial infection and is a requisite for the accumulation of suberin polyaliphatics which provide the barrier to fungal infection. Results showed that the inhibitor treatments that caused changes in polyamine content generally did not influence wound-induced accumulation of SPP. Such lack of correlation was found for inhibitors involved in metabolism and oxidation of putrescine (arginine decarboxylase, ornithine decarboxylase, and diamine oxidase). However, accumulation of SPP was dramatically reduced by treatment with guazatine, a potent inhibitor of polyamine oxidase (PAO), and methylglyoxal-bis(guanylhydrazone), a putative inhibitor of S-adenosylmethione decarboxylase which may also cross-react to inhibit PAO. The mode of action of these inhibitors is presumed to be blockage of essential H2O2 production within the WH cell wall. These results are of great importance in understanding the mechanisms modulating WH and ultimately controlling related infections and associated postharvest losses.

Breast mass segmentation in ultrasound with selective kernel U-Net convolutional neural network.

In this work, we propose a deep learning method for breast mass segmentation in ultrasound (US). Variations in breast mass size and image characteristics make the automatic segmentation difficult. To address this issue, we developed a selective kernel (SK) U-Net convolutional neural network. The aim of the SKs was to adjust network's receptive fields via an attention mechanism, and fuse feature maps extracted with dilated and conventional convolutions. The proposed method was developed and evaluated using US images collected from 882 breast masses. Moreover, we used three datasets of US images collected at different medical centers for testing (893 US images). On our test set of 150 US images, the SK-U-Net achieved mean Dice score of 0.826, and outperformed regular U-Net, Dice score of 0.778. When evaluated on three separate datasets, the proposed method yielded mean Dice scores ranging from 0.646 to 0.780. Additional fine-tuning of our better-performing model with data collected at different centers improved mean Dice scores by ~6%. SK-U-Net utilized both dilated and regular convolutions to process US images. We found strong correlation, Spearman's rank coefficient of 0.7, between the utilization of dilated convolutions and breast mass size in the case of network's expansion path. Our study shows the usefulness of deep learning methods for breast mass segmentation. SK-U-Net implementation and pre-trained weights can be found at github.com/mbyr/bus_seg.

Preparing Students for Success: Differential Outcomes by Preschool Experience in Baltimore City, Maryland.

An early adopter of public preschool (i.e., pre-kindergarten, "pre-k"), evidence from Baltimore City, Maryland, can provide insight for those working to improve access to early education opportunities. We followed a cohort of children entering kindergarten in Baltimore City Public Schools during the 2007-2008 year through the 2010-2011 academic year. Students were grouped by pre-k experience: public pre-k (n = 2828), Head Start (n = 839), Head Start plus public pre-k (n = 247), private pre-k (n = 993), or informal care (n = 975). After adjusting for individual- and school-level characteristics, students from the Head Start plus public pre-k group were the most likely to enter kindergarten with the foundational skills and behaviors needed to be successful (vs. all groups, P ≤ .001). Students in informal care were the least likely to enter kindergarten with this skillset (vs. all pre-k groups P ≤ .001). Children from informal care were also significantly more likely than all other groups to be chronically absent in kindergarten (P ≤ .001). By third grade, children from informal care were least likely to be reading on grade level and most likely to have been retained a grade (vs. all pre-k groups P ≤ .001). Children from disadvantaged populations who were not enrolled in pre-k faced significant difficulties keeping up with their peers throughout elementary school; interventions to improve their transition to school and increase their likelihood of academic success are warranted. Universal preschool is likely to improve education outcomes for children in urban areas.

Evaluation of activity and potential for development of antimicrobial resistance to a new tinted 2% chlorhexidine gluconate/70% isopropyl alcohol film-forming sterile preoperative skin preparation.

OBJECTIVES: Chlorhexidine gluconate (CHG) is routinely used for skin antisepsis before surgery. Its activity may be affected by formulation ingredients and the presence of organic matter such as blood and proteins. This in vitro study was designed to evaluate the antimicrobial activity of a new CHG skin prep containing a film-forming copolymer, and detect its potential for developing resistance and the potential for cross-resistance to antibiotics after CHG exposure. METHODS: Antimicrobial activity was evaluated in the presence and absence of serum in an in vitro time-kill study. Emergence of resistance to CHG and cross-resistance with antibiotic procedures were performed in vitro using 10 repository isolates from eight species and eight clinical isolate strains equal to the repository isolate strains (four isolates, two resistant and two non-resistant per species). RESULTS: A 5 log10 reduction (99.999%) for all organisms was observed using the copolymer formulation. The activity remained unchanged in the presence of serum. The minimum inhibitory concentration (MIC) did not increase for any of the strains evaluated for emergence of resistance. In addition, there was no change in MIC related to cross-resistance observed for any of the organism/antibiotic combinations tested. CONCLUSIONS: These results suggest that the film-forming copolymer and the tint in the new CHG skin prep did not interfere with antimicrobial efficacy, even in the presence of an organic soil load, and that the tested formulations showed no potential for developing resistance or cross-resistance with antibiotics.

Breast mass classification in sonography with transfer learning using a deep convolutional neural network and color conversion.

PURPOSE: We propose a deep learning-based approach to breast mass classification in sonography and compare it with the assessment of four experienced radiologists employing breast imaging reporting and data system 4th edition lexicon and assessment protocol. METHODS: Several transfer learning techniques are employed to develop classifiers based on a set of 882 ultrasound images of breast masses. Additionally, we introduce the concept of a matching layer. The aim of this layer is to rescale pixel intensities of the grayscale ultrasound images and convert those images to red, green, blue (RGB) to more efficiently utilize the discriminative power of the convolutional neural network pretrained on the ImageNet dataset. We present how this conversion can be determined during fine-tuning using back-propagation. Next, we compare the performance of the transfer learning techniques with and without the color conversion. To show the usefulness of our approach, we additionally evaluate it using two publicly available datasets. RESULTS: Color conversion increased the areas under the receiver operating curve for each transfer learning method. For the better-performing approach utilizing the fine-tuning and the matching layer, the area under the curve was equal to 0.936 on a test set of 150 cases. The areas under the curves for the radiologists reading the same set of cases ranged from 0.806 to 0.882. In the case of the two separate datasets, utilizing the proposed approach we achieved areas under the curve of around 0.890. CONCLUSIONS: The concept of the matching layer is generalizable and can be used to improve the overall performance of the transfer learning techniques using deep convolutional neural networks. When fully developed as a clinical tool, the methods proposed in this paper have the potential to help radiologists with breast mass classification in ultrasound.

Cloning, Expression, and Purification of the Glycosylated Transmembrane Protein, Cation-Dependent Mannose 6-Phosphate Receptor, from Sf9 Cells Using the Baculovirus System.

The cation-dependent mannose 6-phosphate receptor (CD-MPR) is a single-pass type I membrane protein. This protein functions to transport lysosomal enzymes displaying phosphomannosyl residues from the Golgi complex and the cell surface to the lysosome. This glycosylated protein contains three disulfide bridges in its 159-residue extracytoplasmic domain. One of the problems with studying eukaryotic membrane proteins is isolating sufficient quantities. Structural studies typically require several hundred milligrams of highly purified protein. Here we present a method to isolate milligram quantities of CD-MPR/Asn81 suitable for structural studies.

Wounding induces changes in cytokinin and auxin content in potato tuber, but does not induce formation of gibberellins.

Cytokinin, auxin and gibberellin contents in resting and wound-responding potato tubers have not been fully determined and coordinated with wound-healing processes. Using a well-defined wound-healing model system, hormone content and expression of genes associated with hormone turnover were determined in tubers following wounding. Changes in hormone content were coordinated with: (I) formation and completion of the wound closing layer (0-5/6 days), and (II) initiation of phellogen and wound periderm formation (∼ 7 days). Quantifiable amounts of biologically active cytokinins (Z, DZ and IP) were not detected in resting or wound-responding tubers. However, the precursor IPA and catabolic product c-ZOG were found in small amounts in resting and wound-responding tubers. Wound-induced activation of cytokinin biosynthesis was suggested by an increase in t-ZR and c-ZR content at 0.5 days and large increases in IPA and c-ZR content by 3 days and throughout 7 days after wounding suggesting roles in II, but little or no role in I. Expression of key genes involved in cytokinin metabolism followed similar profiles with transcripts decreasing through 3 days and then increasing at 5-7 days after wounding. Both free IAA and IAA-Asp were present in resting tubers. While IAA-Asp was no longer present by 3 days after wounding, IAA content nearly doubled by 5 days and was more than 4-fold greater at 7 days compared to that in resting tuber (0 day) suggesting roles in II, but little or no role in I. Gibberellins were not present in quantifiable amounts in resting or wound-responding tubers. These results suggest that bio-active cytokinins are wound-induced, but their residency is temporal and highly regulated. The transient presence of active cytokinins and corresponding increases in IAA content strongly suggest their involvement in the regulation of wound periderm development. The absence of gibberellins indicates that they are not a regulatory component of wound-healing processes.

Medical students' preferences for problem-based learning in relation to culture and personality: a multicultural study.

OBJECTIVE: The aim of this study was to explore positive and negative preferences towards problem-based learning in relation to personality traits and socio-cultural context. METHODS: The study was an anonymous and voluntary cross-sectional survey of medical students (N=449) in hybrid problem-based curricula in Nepal, Norway and North Dakota. Data was collected on gender, age, year of study, cohabitation and medical school. The PBL Preference Inventory identified students' positive and negative preferences in relation to problem-based learning; the personality traits were detected by the NEO Five-Factor Inventory. The determinants of the two kinds of preferences were analyzed by hierarchical multiple linear regressions. RESULTS: Positive preferences were mostly determined by personality; associations were found with the traits Extra-version, Openness to experience, Conscientiousness and Neuroticism; the first three are related to sociability, curiosity and orderliness, the last, to mental health. The learn-ing environments of such curricula may be supportive for some and unnerving for others who score high on Neuroticism. Negative preferences were rather determined by culture, but also, they correlated with Neuroticism and Conscientiousness. Negative preferences were lower among females and students living in symmetrical relationships. Some high on Conscientiousness disliked group work, and the negative correlation with Agreeableness indicated that less sociable students were not predisposed to this kind of learning activity. CONCLUSIONS: Preferences related to problem-based learning were significantly and independently determined both by personality traits and culture. More insights into the nature of students' preferences may guide aspects of curriculum modifications and the daily facilitation of groups.

Crystal Structure and Functional Analyses of the Lectin Domain of Glucosidase II: Insights into Oligomannose Recognition.

N-Glycans are modified as part of a quality control mechanism during glycoprotein folding in the endoplasmic reticulum (ER). Glucosidase II (GII) plays a critical role by generating monoglucosylated glycans that are recognized by lectin chaperones, calnexin and calreticulin. To understand how the hydrolytic activity of GIIα is enhanced by the mannose 6-phosphate receptor (MPR) homology domain (MRH domain) of its ß subunit, we now report a 1.6 Å resolution crystal structure of the MRH domain of GIIß bound to mannose. A comparison of ligand-bound and unbound structures reveals no major difference in their overall fold, but rather a repositioning of side chains throughout the binding pocket, including Y372. Mutation of Y372 inhibits GII activity, demonstrating an important role for Y372 in regulating GII activity. Comparison of the MRH domains of GIIß, MPRs, and the ER lectin OS-9 identified conserved residues that are critical for the structural integrity and architecture of the carbohydrate binding pocket. As shown by nuclear magnetic resonance spectroscopy, mutations of the primary binding pocket residues and adjacent W409, all of which inhibit the activity of GII both in vitro and in vivo, do not cause a significant change in the overall fold of the GIIß MRH domain but impact locally the stability of the binding pocket. W409 does not directly contact mannose; rather, its indole ring is stabilized by binding into a hydrophobic pocket of an adjacent crystallographic neighbor. This suggests that W409 interacts with a hydrophobic region of the GIIß or GIIα subunit to modulate its effect on GII activity.