Bactericidal human monoclonal antibody 1B1 shows specificity for meningococcal factor H binding protein variant 2 and displaces human factor H.

Thousands of disease cases and hundreds of deaths occur globally each year due to invasive meningococcal disease. Neisseria meningitidis serogroup B (MenB) is the leading cause of such disease in developed countries. Two vaccines, 4CMenB and MenB-fHbp, that protect against MenB are available and include one or two forms respectively of factor H binding protein (fHbp), a key protective antigen. Studies of circulating meningococci have identified over 1380 different fHbp amino acid sequences, which form three immunologically distinct clusters, termed variants 1, 2, and 3. Neither of the current vaccines contains a variant 2 antigen, which is less well characterized than fHbp variants 1 and 3. We characterized the interaction of fHbp variant 2 with humAb 1B1 using biochemical methods and live meningococcal assays. Further, we determined the crystal structure of the complex at 2.4 Å resolution, clearly revealing the epitope and providing the first detailed report of an antibody with distinct specificity for fHbp variant 2. Extensive mutagenesis and binding studies elucidated key hotspots in the interface. This combination of structural and functional studies provides a molecular explanation for the bactericidal potency and specificity of humAb 1B1 for fHbp variant 2. Our studies, focused on fHbp variant 2, expand the understanding of this previously under characterized group of the vast family of variants of fHbp, a virulence factor present on all meningococci. Moreover, the definition of a protective conformational epitope on fHbp variant 2 may support the design and development of novel variant 2-containing MenB vaccines affording greater breadth of protection.

Substrate specificity modification of paraben hydrolase and tannase from Aspergillus oryzae.

Paraben hydrolase and tannase catalyze the hydrolysis of parabens (4-hydroxybenzoic acid esters) and gallic acid (3,4,5-trihydroxybenzoic acid) esters, respectively. Paraben hydrolase (AoPrbA) and tannase (AoTanB) from Aspergillus oryzae belong to the tannase family in the ESTHER database. However, the substrate specificities of AoPrbA and AoTanB are narrow. Based on structural information of Aspergillus niger tannase (PDB code 7k4o), we constructed five single variants of AoPrbA (Thr200Glu, Phe231Gln, Leu232Gln, Ile361Tyr, and Leu428Ser) and four of AoTanB (Glu203Asp, Glu203Thr, His237Ala, and Ser440Leu) to investigate substrate discrimination between AoPrbA and AoTanB. Each variant was expressed in Pichia pastoris and were purified from the culture supernatant. Five purified variants of AoPrbA and four variants of AoTanB showed reduced paraben hydrolase and tannase activities compared with AoPrbA and AoTanB wild types, respectively. Interestingly, the AoPrbA wild type did not hydrolyze gallic acid methyl ester, whereas the Thr200Glu, Leu232Gln, and Leu428Ser variants did, indicating that these three variants acquired tannase activity. In particular, the Leu428Ser variant exhibited considerably greater hydrolysis of gallic acid and protocatechuic acid methyl esters. Meanwhile, the AoTanB wild type, and Glu203Asp, His237Ala and Ser440Leu variants hydrolyzed the protocatechuate methyl and 4-hydroxybenzoate ethyl esters; however, the Glu203Thr variant did not hydrolyze above-mentioned substrates. Additionally, the ratio of paraben hydrolase activity to tannase activity in Ser440Leu was markedly elevated.

The construction of ofloxacin detection in fish matrix based on a shark-derived single-domain antibody.

BACKGROUND: Due to the serious issue of ofloxacin (OFL) abuse, there is an increasingly urgent need for accurate and rapid detection of OFL. Immunoassay has become the "golden method" for detecting OFL in complex matrix beneficial to its applicability for a large-scale screening, rapidity, and simplicity. However, traditional antibodies used in immunoassay present challenges such as time-consuming preparation, unstable sensitivity and specificity, and difficulty in directional evolution. In this paper, we successfully developed an OFL detection method based on a shark-derived single-domain antibody (ssdAb) to address these issues. RESULTS: Using phage display technology and a heterologous expression system, OFL-specific clones 1O11, 1O13, 1O17, 1O19, 1O21, and 2O26 were successfully isolated and expressed in soluble form. Among all OFL-specific ssdAbs, the 1O17 ssdAb exhibited the highest binding affinity to OFL in a concentration-dependence manner. The limit of detection (IC10) of 1O17 ssdAb was calculated as 0.34 ng/mL with a detection range of 3.40-1315.00 ng/mL, and its cross reactivity with other analogs was calculated to be less than 5.98 %, indicating high specificity and sensitivity. Molecular docking results revealed that 100Trp and 101Arg located in the CDR3 region of 1O17 ssdAb were crucial for OFL binding. In fish matrix performance tests, the 1O17 ssdAb did not demonstrate severe matrix interference in OFL-negative fish matrix, achieving satisfactory recovery rates ranging from 83.04 % to 108.82 % with high reproducibility. SIGNIFICANCE: This research provides a new and efficient OFL detection recognition element with significant potential in immunoassay applications, broadening the application scenarios of ssdAbs. It offers valuable insights into the structure-activity relationship between ssdAbs and small molecules, laying a theoretical foundation for the further directional modification and maturation of ssdAbs in subsequent applications.

C-reactive protein and neutrophil-to-lymphocyte ratio as key inflammatory indicators in the diagnosis of cervical necrotizing fasciitis.

BACKGROUND: Cervical necrotizing fasciitis (CNF) is a life-threatening bacterial infection with a diagnostic challenge. Currently, there is insufficient evidence on the diagnostic accuracy of inflammatory indicators in CNF. OBJECTIVE: This study aims to identify key inflammatory indicators and assess their diagnostic accuracy for CNF. METHODS: A diagnostic case-control study was conducted at a tertiary healthcare facility from January 2020 to December 2023. Laboratory data from patients with CNF and non-CNF at admission were evaluated. Key inflammatory indicators were identified through consistent outcomes from multivariable logistic regression and receiver operating characteristic curves analyses. The diagnostic accuracy of these indicators, with the results of combined tests, were calculated. RESULTS: CNF was confirmed in 21 of the 67 patients investigated. C-reactive protein (CRP) and neutrophil-to-lymphocyte ratio (NLR) were identified as key inflammatory indicators, with sensitivities of 0.905 and 0.810, and specificities of 0.870 and 0.913, respectively, at CRP threshold of 165.0 mg/L and NLR of 15.8. Combining CRP and NLR in parallel and serial tests increased sensitivity to 0.952 and specificity to 1.0, respectively. CONCLUSIONS AND SIGNIFICANCE: CRP and NLR have been verified as key inflammatory indicators with satisfactory diagnostic abilities for CNF diagnosis, providing a strong foundation for future studies.

Structure-Assisted Design of Chitosanase Product Specificity for the Production of High-Degree Polymerization Chitooligosaccharides.

Chitosanases are valuable enzymatic tools in the food industry for converting chitosan into functional chitooligosaccharides (COSs). However, most of the chitosanases extensively characterized produced a low degree of polymerization (DP) COSs (DP = 1-3, LdpCOSs), indicating an imperative for enhancements in the product specificity for the high DP COS (DP >3, HdpCOSs) production. In this study, a chitosanase from Methanosarcina sp. 1.H.T.1A.1 (OUC-CsnA4) was cloned and expressed. Analysis of the enzyme-substrate interactions and the subsite architecture of the OUC-CsnA4 indicated that a Ser49 mutation could modify its interaction pattern with the substrate, potentially enhancing product specificity for producing HdpCOSs. Site-directed mutagenesis provided evidence that the S49I and S49P mutations in OUC-CsnA4 enabled the production of up to 24 and 26% of (GlcN)5 from chitosan, respectively─the wild-type enzyme was unable to produce detectable levels of (GlcN)5. These mutations also altered substrate binding preferences, favoring the binding of longer-chain COSs (DP >5) and enhancing (GlcN)5 production. Furthermore, molecular dynamics simulations and molecular docking studies underscored the significance of +2 subsite interactions in determining the (GlcN)4 and (GlcN)5 product specificity. These findings revealed that the positioning and interactions of the reducing end of the substrate within the catalytic cleft are crucial factors influencing the product specificity of chitosanase.

Several orphan solute carriers functionally identified as organic cation transporters: substrates specificity compared with known cation transporters.

Organic cations comprise a significant part of medically relevant drugs and endogenous substances. Such substances need organic cation transporters (OCT) for efficient transfer via cell membranes. However, the membrane transporters of most natural or synthetic organic cations the membrane transporters are still unknown. To identify these transporters, genes of 10 known OCTs and 18 orphan solute carriers (SLC) were overexpressed in HEK293 cells and characterized concerning their transport activities with a broad spectrum of low molecular weight substances emphasizing organic cations. Several SLC35 transporters and SLC38A10 significantly enhanced the transport of numerous relatively hydrophobic organic cations. Significant organic cation transport activities have been found in gene families classified as transporters of other substance classes. For instance, SLC35G3 and SLC38A10 significantly accelerated the uptake of several cations, such as clonidine, 3,4-methylenedioxymethamphetamine, and nicotine, which are known as substrates of a thus far genetically unidentified proton/organic cation antiporter. The transporters SLC35G4 and SLC35F5 stood out by their significantly increased choline uptake, and several other SLC transported choline together with a broader spectrum of organic cations. Overall, there are many more polyspecific organic cation transporters than previously estimated. Several transporters had one predominant substrate but accepted some other cationic substrates, and others showed no particular preference for one substrate but transported several organic cations. The role of these transporters in biology and drug therapy remains to be elucidated.

An evaluation of a lateral flow rapid diagnostic test for Strongyloides stercoralis infection.

Strongyloides stercoralis in humans often presents as a chronic asymptomatic infection. Diagnosis can be challenging due to the limited sensitivity of faecal-based parasitological techniques. A prototype lateral flow rapid diagnostic test (RDT) for the detection of specific antibodies against Strongyloides stercoralis (SsRapid) was evaluated using 143 samples from the serum bank of the Swiss Tropical and Public Health Institute. Group 1 (n=30) comprised serum samples from larvae-positive individuals; the RDT's diagnostic sensitivity was 97% (29/30). Group II comprised serum samples from patients with other parasitic infections (n=86) and Swiss blood donors (n=27); the RDT's diagnostic specificity for this group was 90% (102/113). The RDT showed good diagnostic performance and is a promising point-of-care test for detecting human Strongyloides stercoralis infection.

Can isometric testing substitute for the one repetition maximum squat test?

When measuring maximum strength, a high accuracy and precision is required to monitor the training adaptations. Based on available reliability parameters, the literature suggests the replacement of the one repetition maximum (1RM) by isometric testing to save testing time. However, from a statistical point of view, correlation coefficients do not provide the required information when aiming to replace one test by another. Therefore, the literature suggests the inclusion of the mean absolute error (MAE), the mean absolute percentage error (MAPE) for agreement analysis. Consequently, to check the replaceability of 1RM testing methods, the current study examined the agreement of isometric and dynamic testing methods in the squat and the isometric mid-thigh pull. While in accordance with the literature, correlations were classified high r = 0.638-0.828 and ICC = 0.630-0.828, the agreement analysis provided MAEs of 175.75-444.17 N and MAPEs of 16.16-57.71% indicating an intolerable high measurement error between isometric and dynamic testing conditions in the squat and isometric mid-thigh pull. In contrast to previous studies, using MAE, MAPE supplemented by CCC and BA analysis highlights the poor agreement between the included strength tests. The recommendation to replace 1RM testing with isometric testing routines in the squat does not provide suitable concordance and is not recommended.

Case report: Possible role of low-dose PEM for avoiding unneeded procedures associated with false-positive or equivocal breast MRI results.

Contrast-enhanced breast magnetic resonance imaging (MRI) is currently recommended as a screening tool for high-risk women and has been advocated for women with radiologically dense breast tissue. While breast MRI is acknowledged for its high sensitivity (with an exception for lower-grade ductal carcinoma in situ (DCIS) where emerging techniques like diffusion-weighted imaging offer improvement), its limitations include sensitivity to hormonal changes and a relatively high false-positive rate, potentially leading to overdiagnosis, increased imaging uncertainty, and unnecessary biopsies. These factors can exacerbate patient anxiety and impose additional costs. Molecular imaging with breast-targeted Positron Emission Tomography (PET) has shown the capability to detect malignancy independent of breast density and hormonal changes. Furthermore, breast-targeted PET has shown higher specificity when compared with MRI. However, traditional PET technology is associated with high radiation dose, which can limit its widespread use particularly in repeated studies or for undiagnosed patients. In this case report, we present a clinical application of low-dose breast imaging utilizing a breast-targeted PET camera (Radialis PET imager, Radialis Inc). The case involves a 33-year-old female patient who had multiple enhanced lesions detected on breast MRI after surgical removal of a malignant phyllodes tumor from the right breast. A benign core biopsy was obtained from the largest lesion seen in the left breast. One month after the MRI, 18F-fluorodeoxyglucose (18F-FDG) PET imaging session was performed using the Radialis PET Imager. Although the Radialis PET Imager has proven high count sensitivity and the capability to detect breast lesions with low metabolic activity (at a dose similar to mammography), no areas of increased 18F-FDG uptake were visualized in this particular case. The patient underwent a right-sided nipple-sparing mastectomy and left-sided lumpectomy, with bilateral reconstruction. The excised left breast tissue was completely benign, as suggested by both core biopsy and the PET results. The case presented highlights a promising clinical application of low-dose breast-targeted PET imaging to mitigate the uncertainty associated with MRI while keeping radiation doses within the safe range typically used in X-ray mammography.

Application of the international system for reporting serous fluid cytopathology on pleural effusion cytology with paired pleural biopsy: A new insight and novel approach on risk of malignancy.

INTRODUCTION: The risk of malignancy (ROM) remains an area of interest for further evaluation in reporting systems including in International System for reporting serous fluid cytopathology (TIS), which is a standardized system for reporting effusion cytology. Herein, we report our findings in further investigation of ROM in TIS by studying on paired pleural effusion specimens and corresponding pleural biopsies with emphasis on negative for malignancy, and atypia of undetermined significance categories. MATERIALS AND METHODS: The  Johns Hopkins Hospital pathology database was retrospectively searched for patients with a pleural biopsy (PBX) and a paired pleural effusion (PF) cytology specimens over a 4-year period. We employed the TIS categories. The following statistical parameters were evaluated: sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and ROM. RESULTS: A total of 223 patient cases were included. Effusions TIS reclassification and ROM were as follows: 1.8% non-diagnostic (ROM 75%), 75.8% negative for malignancy (ROM 23%), 4.9% atypical cells of undetermined significance (ROM 45%), 2.2% suspicious for malignancy (ROM 80%), and 15.2% malignant (ROM 100%). Overall accuracy, sensitivity, specificity, PPV and NPV were calculated and were 79.4%, 45%, 97.7%, 91.2% and 77%, respectively. Among, discordant cases diagnosed negative for malignancy on PF and positive for malignancy on PBX, there were significant number of lymphomas, mesotheliomas, and sarcomas. Lung cancer was the most common carcinoma; however, rare types of carcinomas were noted. Cells blocks and immunohistochemistry (IHC) studies were utilized to confirm either malignant conditions or rule out malignancy in both cell blocks and histology biopsies. CONCLUSION: This study demonstrates the high specificity and ROM for 'malignant' and 'suspicious for malignancy' categories in the TIS reporting system and highlights the modest negative predictive value for the 'negative for malignancy' category. Although Tissue biopsies are usually considered as 'gold standard', any definitive diagnosis of malignancy of body fluid should be considered positive for malignancy in further clinical decision-making.

Support Enzyme Loading Influences the Effect of Aldehyde Dextran Modification on the Specificity of Immobilized Ficin for Large Proteins.

It has been reported that the modification of immobilized glyoxyl-ficin with aldehyde dextran can promote steric hindrances that greatly reduce the activity of the immobilized protease against hemoglobin, while the protease still maintained a reasonable level of activity against casein. In this paper, we studied if this effect may be different depending on the amount of ficin loaded on the support. For this purpose, both the moderately loaded and the overloaded glyoxyl-ficin biocatalysts were prepared and modified with aldehyde dextran. While the moderately loaded biocatalyst had a significantly reduced activity, mainly against hemoglobin, the activity of the overloaded biocatalyst was almost maintained. This suggests that aldehyde dextran was able to modify areas of the moderately loaded enzyme that were not available when the enzyme was overloaded. This modification promoted a significant increase in biocatalyst stability for both biocatalysts, but the stability was higher for the overloaded biocatalyst (perhaps due to a combination of inter- and intramolecular crosslinking).

Tailoring the specificity of ficin versus large hemoglobin and small casein by co-immobilizing inert proteins on the immobilized enzyme layer and further modification with aldehyde dextran.

Ficin has been immobilized at full loading on glyoxyl agarose beads. Then, ficin was blocked with 2,2'-dipyridyldisulfide. To be effective, the modification must be performed in the presence of 0.5 M urea, as the enzyme was not inhibited under standard conditions, very likely because the catalytic Cys was not fully exposed to the medium. Activity could be fully recovered by incubation with 1 M mercaptoethanol. This biocatalyst could hydrolyze hemoglobin and casein. The objective of this paper was to increase the enzyme specificity versus small proteins by generating steric hindrances to the access of large proteins. The step by step blocking via ionic exchange of the biocatalyst with aminated bovine serum albumin (BSA), aldehyde dextran and a second layer of aminated BSA produced a biocatalyst that maintained its activity versus small synthetic substrates, increased the biocatalyst stability, while reduced its activity to over 50 % versus casein. Interestingly, this treatment almost fully annulled the activity versus hemoglobin, more effectively at 37 °C than at 55 °C. The biocatalyst could be reused 5 times without changes in activity. The changes could be caused by steric hindrances, but it cannot be discarded some changes in enzyme sequence specificity caused by the modifications.

Epiphytic bacterial community composition on four submerged macrophytes in different regions of Taihu Lake.

The epiphytic bacteria in aquatic ecosystems, inhabiting a unique ecological niche with significant ecological function, have long been the subject of attention. Habitat characteristics and plant species are believed to be important in controlling the assembly of epiphytic bacteria. However, the underlying principle governing the assembly of the epiphytic bacterial community on macrophytes is far from clear. In this study, we systematically compared the diversity and community composition of epiphytic bacteria both in different habitats and on different species of macrophytes where they were attached. Results suggested that neither the plant species nor the habitat had a significant effect on the diversity and community of epiphytic bacteria independently, indicating that the epiphytic bacterial community composition was correlated to both geographical distance and individual species of macrophytes. Furthermore, almost all of the abundant taxa were shared between different lake regions or macrophyte species, and the most abundant bacteria belonged to Proteobacteria and Firmicutes. Our results demonstrated that the competitive lottery model may explain the pattern of epiphytic bacterial colonization of submerged macrophyte surfaces. This research could provide a new perspective for exploring plant-microbe interaction in aquatic systems and new evidence for the lottery model as the mechanism best explaining the assembly of epiphytic bacteria.

Performance of a Tic Screening Tool (MOVeIT) in Comparison to Expert Clinician Assessment in a Developmental-Behavioral Pediatrics Clinic Sample.

Youth with intellectual and developmental disabilities typically have higher rates of tics and stereotypies compared to children with otherwise typical development. Differentiating between these two pediatric movement disorders can be challenging due to overlapping clinical features, but is relevant due to distinct treatment modalities. The current study evaluated sensitivity and specificity of a tic screening measure, the Motor or Vocal Inventory of Tics (MOVeIT) in a pediatric sample enriched for stereotypy and tics. Children (n=199, age 2-15 years old) receiving care in a developmental-behavioral pediatrics clinic underwent a gold-standard diagnostic assessment by a tic expert; these evaluations were compared to the MOVeIT. The MOVeIT demonstrated good sensitivity (89.8%) and relatively lower specificity (57.1%) compared to tic expert for detecting tics in the overall sample. Specificity of the MOVeIT to identify tics improved to 75% when excluding children with co-occurring stereotypy. For children with tics and co-occurring stereotypy, sensitivity remained high (91.9%) but specificity was low (39.1%). The area under the curve (AUC) value to detect tics on the MOVeIT compared to the tic expert gold standard was significantly higher for children without stereotypy (AUC=85.7%) than those with stereotypy (AUC=64.3%, p <0.01). Overall, the ability to detect tics was better in those without co-occurring stereotypy symptoms. Further work is needed to establish the utility of the MOVeIT in populations where there is a high likelihood of co-occurring tics and stereotypy and in general population settings. Accurate distinction between tics and stereotypy will guide choices for intervention and anticipatory guidance for families.

Imperfect symmetry facilitated the evolution of specificity and high-order stoichiometry in vertebrate hemoglobin.

Many proteins form paralogous multimers - molecular complexes in which evolutionarily related proteins are arranged into specific quaternary structures. Little is known about the mechanisms by which they acquired their stoichiometry (the number of total subunits in the complex) and heterospecificity (the preference of subunits for their paralogs rather than other copies of the same protein). Here we use ancestral protein reconstruction and biochemical experiments to study historical increases in stoichiometry and specificity during the evolution of vertebrate hemoglobin (Hb), a α2ß2 heterotetramer that evolved from a homodimeric ancestor after a gene duplication. We show that the mechanisms for this evolutionary transition was simple. One hydrophobic substitution in subunit ß after the gene duplication was sufficient to cause the ancestral dimer to homotetramerize with high affinity across a new interface. During this same interval, a single-residue deletion in subunit α at the older interface conferred specificity for the heterotetrameric form and the trans-orientation of subunits within it. These sudden transitions in stoichiometry and specificity were possible because the interfaces in Hb are isologous - involving the same surface patch on interacting subunits, rotated 180° relative to each other - but the symmetry is slightly imperfect. This architecture amplifies the impacts of individual mutations on stoichiometry and specificity, especially in higher-order complexes, and allows single substitutions to differentially affect heteromeric vs homomeric interactions. Many multimers are isologous, and symmetry in proteins is always imperfect; our findings therefore suggest that elaborate and specific molecular complexes may often evolve via simple genetic and physical mechanisms.

Predictive signature of murine and human host response to typical and atypical pneumonia.

BACKGROUND: Pneumonia due to typical bacterial, atypical bacterial and viral pathogens can be difficult to clinically differentiate. Host response-based diagnostics are emerging as a complementary diagnostic strategy to pathogen detection. METHODS: We used murine models of typical bacterial, atypical bacterial and viral pneumonia to develop diagnostic signatures and understand the host's response to these types of infections. Mice were intranasally inoculated with Streptococcus pneumoniae, Mycoplasma pneumoniae, influenza or saline as a control. Peripheral blood gene expression analysis was performed at multiple time points. Differentially expressed genes were used to perform gene set enrichment analysis and generate diagnostic signatures. These murine-derived signatures were externally validated in silico using human gene expression data. The response to S. pneumoniae was the most rapid and robust. RESULTS: Mice infected with M. pneumoniae had a delayed response more similar to influenza-infected animals. Diagnostic signatures for the three types of infection had 0.94-1.00 area under the receiver operator curve (auROC). Validation in five human gene expression datasets revealed auROC of 0.82-0.96. DISCUSSION: This study identified discrete host responses to typical bacterial, atypical bacterial and viral aetiologies of pneumonia in mice. These signatures validated well in humans, highlighting the conserved nature of the host response to these pathogen classes.

Development of cognitive frailty screening tool among community-dwelling older adults.

Purpose: To develop a brief screening tool consisting of twelve items that can be self-administered for rapid identification of older adults at risk of cognitive frailty (CF), named as Cognitive Frailty Screening Tool (CFST). Patients and methods: A total of 1318 community-dwelling individuals aged 60 years and above were selected and assessed for cognitive frailty using a set of neuropsychology batteries and physical function tests. A binary logistic regression (BLR) was used to identify predictors of CF to be used as items in the screening tool. A suitable cut-off point was developed using receiver operating characteristic analysis. Results: Twelve items were included in the screening tool, comprising of gender, education years, medical history, depressive symptoms and functional status as well as lifestyle activities. The area under the curve (AUC) was 0.817 (95 % CI:0.774-0.861), indicating an excellent discriminating power. The sensitivity and specificity for cut-off 7 were 80.8 % and 79.0 %, with an acceptable range of positive predictive value (PPV) (73.3 %) and negative predictive value (NPV) (85.2 %) for screening tools. Concurrent validity of CFST score with standard cognitive and frailty assessment tools shows a significant association with the total score of CFST with low to moderate correlation (p < 0.05 for all parameters). Conclusion: CFST had good sensitivity and specificity and was valid for community-dwelling older adults. There is a need to evaluate further the cost-effectiveness of implementing CFST as a screening for the risk of CF in the community. Its usage in clinical settings needs further validation.

Deep-learning-based design of synthetic orthologs of SH3 signaling domains.

Evolution-based deep generative models represent an exciting direction in understanding and designing proteins. An open question is whether such models can learn specialized functional constraints that control fitness in specific biological contexts. Here, we examine the ability of generative models to produce synthetic versions of Src-homology 3 (SH3) domains that mediate signaling in the Sho1 osmotic stress response pathway of yeast. We show that a variational autoencoder (VAE) model produces artificial sequences that experimentally recapitulate the function of natural SH3 domains. More generally, the model organizes all fungal SH3 domains such that locality in the model latent space (but not simply locality in sequence space) enriches the design of synthetic orthologs and exposes non-obvious amino acid constraints distributed near and far from the SH3 ligand-binding site. The ability of generative models to design ortholog-like functions in vivo opens new avenues for engineering protein function in specific cellular contexts and environments.

Delving into human α1,4-galactosyltransferase acceptor specificity: The role of enzyme dimerization.

Human α1,4-galactosyltransferase (A4galt), a Golgi apparatus-resident GT, synthesizes Gb3 glycosphingolipid (GSL) and P1 glycotope on glycoproteins (GPs), which are receptors for Shiga toxin types 1 and 2. Despite the significant role of A4galt in glycosylation processes, the molecular mechanisms underlying its varied acceptor specificities remain poorly understood. Here, we attempted to elucidate A4galt specificity towards GSLs and GPs by exploring its interaction with GTs with various acceptor specificities, GP-specific ß1,4-galactosyltransferase 1 (B4galt1) and GSL-specific ß1,4-galactosyltransferase isoenzymes 5 and 6 (B4galt5 and B4galt6). Using a novel NanoBiT assay, we found that A4galt can form homodimers and heterodimers with B4galt1 and B4galt5 in two cell lines, human embryonic kidney cells (HEK293T) and Chinese hamster ovary cells (CHO-Lec2). We found that A4galt-B4galts heterodimers preferred N-terminally tagged interactions, while in A4galt homodimers, the favored localization of the fused tag depended on the cell line used. Furthermore, by employing AlphaFold for state-of-the-art structural prediction, we analyzed the interactions and structures of these enzyme complexes. Our analysis highlighted that the A4galt-B4galt5 heterodimer exhibited the highest prediction confidence, indicating a significant role of A4galt heterodimerization in determining enzyme specificity toward GSLs and GPs. These findings enhance our knowledge of A4galt acceptor specificity and may contribute to a better comprehension of pathomechanisms of the Shiga toxin-related diseases.