Cold Spot SCANNER: Colab Notebook for predicting cold spots in protein-protein interfaces.

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 .

ProBASS - a language model with sequence and structural features for predicting the effect of mutations on binding affinity.

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 (ΔΔGbind) 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 ΔΔGbind. 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 ΔΔGbind values. Our model, ProBASS (Protein Binding Affinity from Structure and Sequence) achieved a correlation with experimental ΔΔGbind 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 ΔΔGbind values across multiple PPIs to refine the pre-trained PLMs represents a successful approach for achieving a precise and broadly applicable model for ΔΔGbind prediction, greatly facilitating future protein engineering and design studies.

Protective measures, personal experience, and the affective psychology of time.

We examined the role of time and affect in intentions to purchase a risk-protective measure (Studies 1 and 2) and explored participant abilities to factor time into the likelihood judgments that presumably underlie such intentions (Study 3). Participants worried more about losing their possessions and were more likely to purchase a protective measure given a longer term lease than a short-term lease, but only if their belongings were described in affect-poor terms. If described instead as being particularly special and affect-rich, participants neglected time and were about equally likely to purchase a risk-protective measure for shorter and longer term leases. However, and consistent with prior literature, the cognitive mechanism underlying this time-neglect-with-affect-richness effect seemed to be the greater use of the affect heuristic in the shorter term than the longer term. Study 2 results demonstrated that prior experience with having been burglarized amplified the interactive effect of time and affect. Greater deliberation did not attenuate this effect as hypothesized whether deliberation was measured through numeracy or manipulated through instructions. The results of Study 3 indicated that few participants are able to calculate correctly the risk numbers necessary to take time into account. Two possible solutions to encourage more purchases of protective measures in the long term are discussed.

Cold spots are universal in protein-protein interactions.

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.

The Influence of Rotational Length, along with Pre- and Post-Grazing Measures on Nutritional Composition of Pasture during Winter and Spring on New Zealand Dairy Farms.

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.

A Nutritional Investigation of Major Feed Types and Feed Rations Used in Medium-Scale Dairy Production Systems in Sri Lanka.

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.

Glucose-regulated protein 78 substrate-binding domain alters its conformation upon EGCG inhibitor binding to nucleotide-binding domain: Molecular dynamics studies.

Glucose-regulated protein 78 (GRP78), is overexpressed in glioblastoma, other tumors and during viral and bacterial infections, and so, it is postulated to be a key drug target. EGCG, an ATP-competitive natural inhibitor, inhibits GRP78 effect in glioblastoma. Structural basis of its action on GRP78 nucleotide-binding domain and selectivity has been investigated. We were interested in exploring the large-scale conformational movements travelling to substrate-binding domain via linker region. Conformational effects of EGCG inhibitor as well as ATP on full length GRP78 protein were studied using powerful MD simulations. Binding of EGCG decreases mobility of residues in SBDα lid region as compared to ATP-bound state and similar to apo state. The decreased mobility may prevent its opening and closing over SBDß. This hindrance to SBDα subdomain movement, in turn, may reduce the binding of substrate peptide to SBDß. EGCG binding folds the protein stably as opposed to ATP binding. Several results from EGCG binding simulations are similar to that of the apo state. Key insights from these results reveal that after EGCG binding upon competitive inhibition with ATP, GRP78 conformation may revert to that of inactive, apo state. Further, SBD may adopt a semi-open conformation unable to facilitate association of substrates.

Tumor necrosis factor inhibiting angiogenesis in vitro.

The effect of recombinant human tumor necrosis factor (TNF) on the capillary growth was evaluated using an in vitro angiogenesis model recently developed. Sprague-Dawley rat microvessel fragments, from epididymal fat pads, seeded onto the confluent culture of myofibroblastic cells from the same tissue origin, gave rise to microvascular networks on and in the multilayered myofibroblastic cells. In contrast, the capillary growth from the vessel fragments was markedly inhibited in the presence of 10-1,000 U TNF/ml. Monoclonal antibody against TNF completely neutralized the capillary growth inhibitory activity of TNF. The mode of angiogenesis inhibitory action of TNF was also examined by use of bovine capillary endothelial (BCE) cells and rat myofibroblastic cells. TNF exerted growth inhibitory and cytotoxic actions against BCE cells cultivated on the various basement membrane components, such as extracellular matrix secreted by BCE cells, fibronectin, laminin, and type IV collagen. An irreversible damage to most of the BCE cells was observed ultrastructurally after 60 hours' exposure to TNF. TNF, however, did not injure the myofibroblastic cells but rather stimulated their growth. These findings indicate that TNF inhibits in vitro capillary growth by its direct cytostatic and cytotoxic actions to microvascular endothelial cells.

Mitomycin C resistance induced by TCF-3 overexpression in gastric cancer cell line MKN28 is associated with DT-diaphorase down-regulation.

TCF transcription factors are mediators of the WNT signaling pathway and are antagonized by the transforming growth factor beta signaling pathway. Here human TCF-3 has been cloned and characterized. Differential expression analyses of TCF genes in gastric cancer revealed that TCF-1 was expressed in most cases of primary gastric cancer at almost the same level as in normal gastric mucosa and that TCF-3 was occasionally up-regulated in primary gastric cancer. The TCF-3 expression vector was transfected to gastric cancer cell line MKN28 to establish stable transformants. Three independent MKN28 transformants overexpressing TCF-3 showed about 8-fold resistance to mitomycin C (MMC; IC50, 2.4 microg/ml) compared with MKN28 vector transfectants (IC50 = 0.3 microg/ml). Among the 10 drug resistance-associated genes examined in this study, the DT-diaphorase (DTD) gene was down-regulated in three MKN28 transformants overexpressing TCF-3. DTD mRNA was also down-regulated in primary gastric cancer with TCF-3 up-regulation. In addition, DTD protein was down-regulated in three MKN28 transformants overexpressing TCF-3 compared with MKN28 vector transfectants. DTD is implicated in the activation of MMC in target cells, and DTD down-regulation explains MMC resistance. MMC resistance induced by TCF-3 overexpression is probably due to DTD down-regulation, which might provide a possible target for new therapy of drug-resistant gastric cancer.

[Establishment of a human lung squamous cell carcinoma cell line LK-17, and characterization of procoagulant, platelet aggregation and metastatic potential].

A squamous cell carcinoma cell line LK-17 was established from original surgical specimen of the lung. Doubling time of LK-17 in vitro is 43.2 hours, and chromosome analysis shows various abnormality and main modeat 62. LK-17 shows stable metastatic potential to the lung of nude mouse when injected i.v. LK-17 cells show platelet aggregating activity with number population dependent manner. LK-17 secretes direct factor X activating procoagulant which differs from those of tissue factor, cystein protease A and coagulant cancer antigen 1. These platelet aggregation potential and procoagulant activity may play a important roll in metastatic process.

[Establishment and characterization of human lung cancer cell line (KUM.LK-2)].

Human lung cancer cell line, named KUM.LK-2 was established from xenograft implanted in nude mice and maintained for 43 months. And serially transplanted in nude mice. This line has the following biological characters. 1) Spontaneous lung metastases are made in nude mice inoculated subcutaneously. 2) This line can produce some kinds of proteases, like as tissue type plasminogen activator, urokinase type plasminogen activator and collagenase. 3) KUM.LK-2 has the ability of platelet aggregation.

Morphometric nerve fiber analysis and aging process of the human inferior alveolar nerve.

We studied morphometric nerve fiber analysis and the aging process of the inferior alveolar nerve (IAN). Human IANs were resected at the mandibular foramen. The preparation of sections involved fixation, washing, dehydration, embedding, sectioning and staining as described in our previous reports. We estimated the total number of myelinated axons in the IAN being average 26,200, the transverse area of those being average 37.1 microns 2, the average perimeter of those being 23.0 microns, the average circularity ratio of those being 0.85, respectively. According to these results, the IAN did not demonstrate notable age-relation change in any measured item. The IAN differed from not only our previous data on the human motor peripheral nerves but also those on the human sensory peripheral nerves. With regard to the aging process, the IAN indicated morphological features compared with other peripheral nerves.

Differentiation of histochemical properties of masticatory muscles in Bovidae and Cervidae (Artiodactyla).

We analyzed the masticatory muscles (masseter, temporal, medial pterygoid and lateral pterygoid muscles) of Bovidae and Cervidae (Artiodactyla) for the histochemical characteristics of their fiber types. Analysis of muscle fiber types in the present study was based on the staining reaction for SDH, Sudan black B, alpha-GPDH, and myosin-ATPase after alkaline preincubations. Histochemical properties were found to contribute to masticatory function, including a slow-twitch fatigue resistant activity derived from the high percentage of red fibers, in spite of the differences in the distributions of fiber types in three portions (superficial, medial and profound portions) of each masticatory muscle. These results indicate a correlation between the histochemical profiles of individual masticatory muscles in these species and their functions during jaw movements.

[Clinical evaluation of second-look operations in colorectal cancer].

The second look operation (SLO) for early detection of recurrent tumor is indicated rarely because of the advance in diagnostic techniques such as imaging radiology, tumor marker serology and colonofiberscopy. The SLO was performed in 21 out of 362 curative cases of colorectal cancer resected in our department; 13 for re-elevation of plasma CEA level (Group 1), 6 for unclear symptoms or signs (Group 2) and 2 for postchemotherapy or postirradiation (Group 3). Liver metastases were found in four cases in group 1 and two in group 2. Nothing was found in three cases in group 1 and two in group 2. In group 3 the radical operation was carried out in all cases following SLO with good survival results. In group 1 and 2 the excisional rate of liver metastases in 57%, its salvage rate 75%, and the excisional rate of the lymph nodes is 50%, with a salvage rate of 100%. The SLO today is indicated for cases of asymptomatic re-elevation of CEA titer, for cases of undeterminable signs or for cases of postchemotherapeutic or postirradiation improvement.

[A case of non-resectable thymoma which responded to the administration of adriamycin, vindesine and 5-FU].

Surgery and radiotherapy have been considered to be the primary therapies for malignant thymoma. However, the results of these therapies for invasive thymoma are poor. Recently, combination chemotherapy for invasive thymoma has been tried and good results have been reported. A 50-year-old female was admitted to our hospital. Her chest film showed a large anterior mediastinal tumor which was diagnosed as invasive thymoma. In order to remove the tumor, median sternotomy and 4th intercostal thoracotomy was done but the mass was found to have invaded to the adjacent tissues, and so only partial resection of the tumor was performed. Postoperatively, the patient was treated with Adriamycin, Vindesine and 5-FU, as a result of which the mass completely disappeared and CR was achieved. Our regimen, AVF, for non-resectable thymoma is therefore thought to be a potentially effective treatment.

[Influence of a fosfomycin combination on the anti-tumor effect of CDDP].

To elucidate the influence of fosfomycin (FOM) on the anti-cancer effect of cisplatin (CDDP), experimental chemotherapy of CDDP with or without FOM was performed. A human lung cancer cell line, HLC-1 serially transplanted into nude mice was treated with CDDP 10 mg/kg (i.p.). Tumor doubling time in CDDP group and CDDP + FOM group are 26.78 +/- 6.3 days and 33.90 +/- 11.82 days respectively, and they are significantly elongated compared to control group. Serum NAG level in CDDP + FOM group was significantly lowered. Therefore, we concluded that anti-tumor effect of CDDP was not diminished by FOM combination.

[Flow cytometric DNA analysis of malignant potential in colorectal carcinoma--DNA ploidy pattern and liver metastasis].

On the purpose of elucidating the malignant potential, flow cytometric DNA analyses were carried out using paraffin-embedded materials of 82 subserosal, serosal and adventitial invasive colorectal carcinoma. The ratio of DNA diploidy against DNA aneuploidy was one to two. DNA ploidy pattern was not correlated with histological grade but with histopathological factors such as lymphatic permeation, lymph node metastasis and venous invasion. At the primary operation, the rate of the liver metastasis in DNA aneuploid cancer was 16.4% but in DNA diploid cancer the liver metastasis was not observed at all. On the liver metastasis, the 5-year disease free survival rates were lower in patients with DNA aneuploidy (72.8%) than those with DNA diploidy (95.2%). These results indicate that DNA ploidy pattern is related to liver metastasis. Furthermore, the 5-year survival rates in patients with absolute curative resection were lower significantly in DNA aneuploidy (62.5%) than DNA diploidy (92.9%). In conclusion flow cytometric DNA analysis is useful for evaluating the biological malignant potential and predicting the liver metastasis in colorectal carcinoma.