Search

Abstract Background Protein–protein interactions (PPIs) are conveyed through binding interfaces or surface patches on proteins that become buried upon binding. Structural and biophysical analysis of many protein–protein interfaces revealed certain unique features of these surfaces that determine the energetics of interactions and play a critical role in protein evolution. One of the significant aspects of binding interfaces is the presence of binding hot spots, where mutations are highly deleterious for binding. Conversely, binding cold spots are positions occupied by suboptimal amino acids and several mutations in such positions could lead to affinity enhancement. While there are many software programs for identification of hot spot positions, there is currently a lack of software for cold spot detection. Results In this paper, we present Cold Spot SCANNER, a Colab Notebook, which scans a PPI binding interface and identifies cold spots resulting from cavities, unfavorable charge-charge, and unfavorable charge-hydrophobic interactions. The software offers a Py3DMOL-based interface that allows users to visualize cold spots in the context of the protein structure and generates a zip file containing the results for easy download. Conclusions Cold spot identification is of great importance to protein engineering studies and provides a useful insight into protein evolution. Cold Spot SCANNER is open to all users without login requirements and can be accessible at: https://colab.research.google.com/github/sagagugit/Cold-Spot-Scanner/blob/main/Cold_Spot_Scanner.ipynb .

In this paper, the nutritional quality, digestibility, and chemical composition of major feed types as well as the use of those feeds in rations by medium-scale dairy farmers in the Kurunegala district of Sri Lanka were studied. Nine dairy farms were visited fortnightly over a five-month period to identify the feeds that were commonly used. All farms operated under a stall-feeding system in which a manually mixed ration (MMR) was fed 2–3 times daily. Four forages were identified: Guinea grass ecotype A (Panicum maximum), called Guinea grass; Hybrid Napier CO-3 (Pennisetum purpureum × Pennisetum americanum), called CO-3 grass; Gliricidia (Gliricidia sepium); and maize stover (Zea mays L.), along with three other supplementary feeds (maize silage, barley distillers’ by-products, and commercially formulated cattle feed). These feeds were subjected to proximate analysis and in vitro digestibility analysis. The metabolisable energy (ME) of the forages ranged from 7.5–10.0 MJ/kg dry matter (DM), with the ME of Guinea grass and CO-3 grass (7.5 and 8.0 MJ/kg DM, respectively) being lower than that of Gliricidia (10.0 MJ/kg DM). The neutral detergent fibre (NDF) concentration of both Guinea grass and CO-3 grass (both 72% DM) was much higher than that of Gliricidia (47% DM). Crude protein (CP) was higher in Gliricidia (17.5% DM) than in either Guinea grass or CO-3 grass (8.0 and 8.8% DM, respectively). The ME of the supplementary feeds varied between 11.0 and 12.8 MJ/kg DM, while CP varied between 15.0 and 24.0% DM. The daily ME intake of cows was consistently 10% lower than their calculated daily energy requirement; for dry cows, the mean intake was 90 MJ/cow/day supplied vs. 101 MJ required, while for cows in early lactation the mean intake was 126 MJ/cow/day supplied vs. 140 MJ required. The average CP intake of lactating cows (13.5% DM) was inadequate (requirements: 16 to 17.5% DM), while the average CP intake of dry cows (11.8% DM) was satisfactory (requirements: 11 to 12% DM). The current study shows that the majority of the feed types used in these medium-scale dairy farms provide insufficient ME or CP to meet the nutritional requirements of either lactating or dry cows irrespective of the quantity of feed provided.

The quality of ryegrass–clover pasture was investigated between August (winter: start of calving) and November (spring: end of breeding) on pasture-based dairy farms (>85% of total feed from pasture) that had short (n = 2, Farms A and B; winter ~30 days, spring ~20–25 days) or long (n = 2, Farms C and D; winter ~35 days, spring ~25–30 days) grazing rotations to determine whether quality was affected by grazing rotation length (RT). Weekly assessments of pasture growth and herbage quality were made using a standardised electronic rising plate meter, and near-infrared spectroscopy, respectively. Data were subjected to repeated measure mixed model analysis, in which herbage quality was the outcome variable. The highest pre-grazing dry matter (PGDM) and height, post-grazing dry matter (DM) and height, and number of live leaves per tiller (leaf regrowth stage, LS) were present in late spring. Neutral detergent fibre (NDF), acid detergent fibre (ADF), metabolisable energy (ME), and organic matter digestibility (OMD) were positively correlated to each other (r2 ≥ 0.8) whilst ADF and lipid, and ADF and OMD were negatively correlated (r2 ≥ −0.8; p < 0.01). Metabolisable energy content was negatively correlated with ADF and NDF (r2 = −0.7, −0.8, respectively), and was inversely related to PGDM. Metabolisable energy was higher (p < 0.05) in farms with shorter (overall mean: 11.2 MJ/kg DM) than longer (10.9 MJ/kg DM) RT. Crude protein was also inversely related to PGDM and was higher with shorter (23.2% DM) than longer (18.3% DM; p < 0.05) RT. Pre-grazing DM affected the amount of pasture that was grazed and, hence, the amount of DM remaining after grazing (post-grazing DM or residual), so that PGDM was correlated with post-grazing height and residual DM (r2 = 0.88 and 0.51, respectively; both p < 0.001). In conclusion, RT, LS, and PGDM during winter and spring influenced the herbage quality, therefore, better management of pastures may enhance the productivity of dairy cows.

Protein-protein interactions (PPIs) govern virtually all cellular processes. Even a single mutation within PPI can significantly influence overall protein functionality and potentially lead to various types of diseases. To date, numerous approaches have emerged for predicting the change in free energy of binding (ΔΔG bind ) resulting from mutations, yet the majority of these methods lack precision. In recent years, protein language models (PLMs) have been developed and shown powerful predictive capabilities by leveraging both sequence and structural data from protein-protein complexes. Yet, PLMs have not been optimized specifically for predicting ΔΔG bind . We developed an approach to predict effects of mutations on PPI binding affinity based on two most advanced protein language models ESM2 and ESM-IF1 that incorporate PPI sequence and structural features, respectively. We used the two models to generate embeddings for each PPI mutant and subsequently fine-tuned our model by training on a large dataset of experimental ΔΔG bind values. Our model, ProBASS (Protein Binding Affinity from Structure and Sequence) achieved a correlation with experimental ΔΔG bind values of 0.83 ± 0.05 for single mutations and 0.69 ± 0.04 for double mutations when model training and testing was done on the same PDB. Moreover, ProBASS exhibited very high correlation (0.81 ± 0.02) between prediction and experiment when training and testing was performed on a dataset containing 2325 single mutations in 132 PPIs. ProBASS surpasses the state-of-the-art methods in correlation with experimental data and could be further trained as more experimental data becomes available. Our results demonstrate that the integration of extensive datasets containing ΔΔG bind values across multiple PPIs to refine the pre-trained PLMs represents a successful approach for achieving a precise and broadly applicable model for ΔΔG bind prediction, greatly facilitating future protein engineering and design studies.

Objective: Detailed quantitative studies on olfaction remain inadequate in patients with paediatric allergic rhinitis (AR). This study examined olfactory dysfunction in children with AR., Methods: From July 2016 to November 2018, children aged 6-9 years were recruited and grouped as AR (n = 30) or without AR (control group, n = 10). Odour identification was evaluated by the Universal Sniff (U-Sniff) test and the Open Essence (OE). The results were compared between the AR and control groups. Intranasal mucosa findings, nasal smear eosinophil counts, blood eosinophil counts, total immunoglobulin E (IgE) levels, Japanese cedar-specific IgE and Dermatophagoides pteronyssinus-specific IgE were evaluated in all participants. Additionally, the presence of sinusitis and adenoid hypertrophy in patients with AR was also evaluated by sinus X-ray examinations., Results: The median U-Sniff test scores were not significantly different between the AR and control groups (9.0 vs. 10.0, respectively; p = 0.107). The OE score was significantly lower in the AR group than in the control group (4.0 vs. 8.0; p = 0.007, respectively), especially in the moderate-to-severe AR group versus the control group (4.0 vs. 8.0; p = 0.004). Furthermore, in the OE, the correct answer rates for 'wood', 'cooking gas' and 'sweaty socks' were significantly lower in the AR group than in the control group., Conclusions: Paediatric AR patients can reduce olfactory identification ability, and the degree may be associated with the severity of AR in nasal mucosal findings. Furthermore, olfactory dysfunction may slow down the response to 'emergency situations', such as gas leak., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023. Published by Elsevier B.V.)

Proteins interact with each other through binding interfaces that differ greatly in size and physico-chemical properties. Within the binding interface, a few residues called hot spots contribute the majority of the binding free energy and are hence irreplaceable. In contrast, cold spots are occupied by suboptimal amino acids, providing possibility for affinity enhancement through mutations. In this study, we identify cold spots due to cavities and unfavorable charge interactions in multiple protein-protein interactions (PPIs). For our cold spot analysis, we first use a small affinity database of PPIs with known structures and affinities and then expand our search to nearly 4000 homo- and heterodimers in the Protein Data Bank (PDB). We observe that cold spots due to cavities are present in nearly all PPIs unrelated to their binding affinity, while unfavorable charge interactions are relatively rare. We also find that most cold spots are located in the periphery of the binding interface, with high-affinity complexes showing fewer centrally located colds spots than low-affinity complexes. A larger number of cold spots is also found in non-cognate interactions compared to their cognate counterparts. Furthermore, our analysis reveals that cold spots are more frequent in homo-dimeric complexes compared to hetero-complexes, likely due to symmetry constraints imposed on sequences of homodimers. Finally, we find that glycines, glutamates, and arginines are the most frequent amino acids appearing at cold spot positions. Our analysis emphasizes the importance of cold spot positions to protein evolution and facilitates protein engineering studies directed at enhancing binding affinity and specificity in a wide range of applications., (© 2022 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Background: Egg allergy is one of the most common food allergies in children. To date, oral immunotherapy (OIT) has been considered as a promising treatment option for egg allergy. However, safety issues remain concerning severe adverse events requiring epinephrine injection. Hence, establishing a safer method to treat egg allergy would be beneficial. We report here two children with egg allergy who were safely treated with sublingual immunotherapy (SLIT) before transitioning to OIT., Case Presentation: Patient 1 was a 7-year-old girl and Patient 2 was a 5-year-old girl. Although OIT for egg had been attempted in both patients, severe anaphylactic symptoms were induced by ingesting only 0.1 g of heated whole egg. Therefore, SLIT was conducted with aqueous suspensions consisting of water and heated whole egg powder. Suspensions were administered sublingually, kept in the mouth for 2 min, and spat out immediately thereafter. SLIT was continued for 7 months for Patient 1 and 8 months for Patient 2 due to the exploratory character of the study. Afterwards, the patients successfully transferred to low-dose OIT with 1 g of heated whole egg (≒170 mg of egg protein) daily, and are continuing the therapy as of June 2020. As for adverse reactions, Patient 1 expressed oral cavity itchiness once at the beginning of SLIT. Patient 2 had no adverse reaction. The levels of antigen-specific IgE decreased in both patients after SLIT, and further decreased after switching to OIT., Conclusions: Few clinical studies have evaluated the efficacy and safety of SLIT for egg allergy. Although the treatment was conducted in only two patients, our results have shown that SLIT is a promising treatment procedure for egg allergy. Further clinical trials will be needed to additionally assess the efficacy and safety of SLIT in children with food allergy.

Although organisms are exposed to various pressure and temperature conditions, information remains limited on how pressure affects biological rhythms. This study investigated how hydrostatic pressure affects the circadian clock (KaiA, KaiB, and KaiC) of cyanobacteria. While the circadian rhythm is inherently robust to temperature change, KaiC phosphorylation cycles that were accelerated from 22 h at 1 bar to 14 h at 200 bars caused the circadian-period length to decline. This decline was caused by the pressure-induced enhancement of KaiC ATPase activity and allosteric effects. Because ATPase activity was elevated in the CI and CII domains of KaiC, while ATP hydrolysis had negative activation volumes (ΔV ≠ ), both domains played key roles in determining the period length of the KaiC phosphorylation cycle. The thermodynamic contraction of the structure of the active site during the transition state might have positioned catalytic residues and lytic water molecules favourably to facilitate ATP hydrolysis. Internal cavities might represent sources of compaction and structural rearrangement in the active site. Overall, the data indicate that pressure differences could alter the circadian rhythms of diverse organisms with evolved thermotolerance, as long as enzymatic reactions defining period length have a specific activation volume.

Background: There are some biomarkers for asthma diagnosis but they are often difficult in clinical use, particularly in pediatric cases. Periostin is an extracellular matrix protein, upregulated in response to IL-4 or IL-13. Serum periostin is expected to be used as a non-invasive biomarker for asthma diagnosis and management., Methods: Twenty-eight children with asthma (BA) and 27 children without asthma (patients with pectus excavatum, etc. as control group) aged 6-16 years were included. Bronchial asthma was diagnosed according to International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire. Fractional exhaled nitric oxide (FeNO), lung function, blood eosinophil counts, total immunoglobulin E (IgE) levels, and serum periostin levels were assessed. Results were compared between BA and controls. Asthma diagnostic accuracies were calculated using receiver operating characteristics (ROC) curve analyses., Results: Serum periostin levels in the BA group were significantly higher than those in the control group [medians (with interquartile ranges), 134.0 (116.3-166.3) vs. 112.0 (97.0-132.0) ng/ml; p = 0.012]. The area under the ROC curve (AUC) for periostin, FeNO, and eosinophil counts were 0.70, 0.72, and 0.84, respectively. After the exclusion of controls with pectus excavatum, AUC for periostin, forced expiratory volume in 1 s (FEV1 ), and maximum mid-expiratory flow rate (MMF) were 0.75, 0.74, and 0.80, respectively., Conclusion: Serum periostin levels were significantly higher in children with asthma. ROC AUC values for periostin were equivalent to conventional biomarkers, including FeNO levels and lung function testing, indicating the utility of serum periostin levels in diagnosing asthma in children., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Purpose: N-acetyl-cysteine (NAC) is a potent antioxidant known to be a precursor of glutathione. The purpose of this study was to investigate the role of NAC in the development of choroidal neovascularization (CNV)., Methods: CNV was induced in C57BL/6 mice by laser photocoagulation of the ocular fundus. Mice were injected intraperitoneally with NAC or vehicle alone. The levels of 4-hydoroxy-2-nonenal (4-HNE)-modified protein and nucleus factor (NF)-κB were determined by wester blotting. The recruitment of macrophages and neutrophils after laser injury was analyzed immunohistochemically and in myeloperoxidase (MPO) assays. Enzyme-linked immunosorbent assays (ELISA) were used to measure monocyte chemotactic protein (MCP)-1, CXCL1, vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR)-1, and VEGFR-2. The extent of CNV was evaluated 7 d after laser injury by lectin staining., Results: In NAC-treated mice with laser-induced injuries, the induction of 4-HNE-modified protein after 3 hr and the activation of NF-κB in nuclear extracts after 6 hr were markedly suppressed compared to vehicle-treated mice. Macrophage and neutrophil recruitment were inhibited and the levels of MCP-1, CXCL1, VEGF, and VEGFR-1 were also lower in NAC-treated mice compared to vehicle-treated mice. Furthermore, the extent of CNV induced was significantly lower in NAC-treated compared to vehicle-treated mice (p = 0.027)., Conclusions: Our results clearly showed that NAC inhibited indicators of oxidative stress and the activation of NF-κB induced by laser injury, and, consequently, suppressed macrophage and neutrophil infiltration and the development of CNV. This suggests novel preventative and interventional therapeutic strategies for age-related macular degeneration.

Background: To compare 12-month results of two single initial treatments--photodynamic therapy with verteporfin alone (PDT group), and this therapy combined with intravitreal bevacizumab (IVB) (COMB group)--for neovascular age-related macular degeneration (AMD), not including patients with polypoidal choroidal vasculopathy (PCV) who were presumed to have AMD., Methods: This retrospective study evaluated 23 eyes in the PDT group and 22 eyes in the COMB group. IVB (1.25 mg) was administered within 2 weeks after PDT. Main outcome measures were best-corrected visual acuity (VA), central foveal thickness by optical coherence tomography, and number of treatments., Results: At month 12, the PDT group had gained 0.7 letter mean VA and the COMB group, 8.8 letters (P = 0.04). Ten eyes (43%) in the PDT group and 19 eyes (86%) in the COMB group received only one treatment, and significant difference was found (P = 0.005). No severe ocular or systemic safety concerns were discovered., Conclusions: Our 12-month results of PDT combined with IVB for Japanese patients with AMD without PCV appeared to be more effective than those of PDT alone with fewer treatments.

How we react to humanitarian crises, epidemics, and other tragic events involving the loss of human lives depends largely on the extent to which we are moved by the size of their associated death tolls. Many studies have demonstrated that people generally exhibit a diminishing sensitivity to the number of human fatalities and, equivalently, a preference for risky (vs. sure) alternatives in decisions under risk involving human losses. However, the reason for this tendency remains unknown. Here we show that the distributions of event-related death tolls that people observe govern their evaluations of, and risk preferences concerning, human fatalities. In particular, we show that our diminishing sensitivity to human fatalities follows from the fact that these death tolls are approximately power-law distributed. We further show that, by manipulating the distribution of mortality-related events that people observe, we can alter their risk preferences in decisions involving fatalities. Finally, we show that the tendency to be risk-seeking in mortality-related decisions is lower in countries in which high-mortality events are more frequently observed. Our results support a model of magnitude evaluation based on memory sampling and relative judgment. This model departs from the utility-based approaches typically encountered in psychology and economics in that it does not rely on stable, underlying value representations to explain valuation and choice, or on choice behavior to derive value functions. Instead, preferences concerning human fatalities emerge spontaneously from the distributions of sampled events and the relative nature of the evaluation process.