Several plant papain-like cysteine proteases are exploited by the food, cosmetic, pharmaceutical and textile industries. However, some of these enzymes can cause allergic reactions. In this context, we investigated the frequency of sensitization and allergic reactions to some fruit and/or latex cysteine proteases, which are used as additives by the food industry to improve and modify the quality of their products. The FABER test was used to analyse the patients‘ sensitization towards five plants and, for comparison, two homologous mite cysteine proteases. In an Italian population of 341 allergic patients, 133 (39%) had IgE specific for at least one of the seven cysteine proteases under investigation. Most of the patients were IgE positive for Der p 1 and/or Der f 1 (96.38%) reported a clinical history suggestive of respiratory allergy to mites, whereas none of the subjects sensitized to the homologs from papaya, pineapple and fig reported allergy symptoms following ingestion of these foods. Only one patient referred symptoms from ingesting kiwifruit. Therefore, the obtained results showed that sensitization to the fruit enzymes was only rarely concomitant with allergic reactions. These observations, together with the literature reports, suggest that the allergy to plant papain-like cysteine proteases might mainly be an occupational disease. Papain-like cysteine proteases (PLCPs) are widespread in nature, having been found in plants, animals, viruses, bacteria, yeasts and protozoa. These enzymes have key physiological functions, including involvement in the regulation of protein turnover in the acidic conditions of lysosomes . PLCPs aThe PLCP protein family includes some well-known proteases, such as actinidin from kiwifruit, papain and chymopapain from papaya fruit and latex, bromelain from pineapple and ficin from fig fruit and latex. In nature, similar to other proteases, to prevent unwanted digestion, they are synthesized as inactive proenzymes containing a N-terminal signal peptide, followed by a propeptide region and a catalytic domain . The prowww.allergome.org, accessed on 26 May 2023) [Carica papaya and is also the first protease for which a crystallographic structure was determined [Papain, named Cari p Papain as an allergen (ay 2023)  is the btermined . It is stermined . In papatermined . Neverthtermined  suggestitermined . These ePlant PLCPs are widely used in the food industry, pharmaceuticals, and cosmetics. For instance, the baking industry exploits these proteases because dough may be prepared more quickly if the gluten has been partially hydrolyzed . In the http://allergen.org/, accessed on 26 May 2023) [Dermatophagoides pteronyssinus and Dermatophagoides farinae, respectively. The list also includes some plant homologous enzymes, such as actinidin, bromelain, and chymopapain, isolated from kiwifruit (Actinidia deliciosa), from the fruit and stem of the pineapple (Ananas comosus), and from the fruit of papaya (Carica papaya), which were registered by the WHO/IUIS with the allergen names Act d 1 [www.allergome.org, accessed on 26 May 2023) from fig (Ficus carica), for which we have here used the names Cari p Papain and Fic c Ficin.Several cysteine proteases of the PLCP family have been reported as a cause of allergic reactions, including the group of mite allergens, such as the major dust-mite allergen Der p 1  and fooday 2023) , isolate Act d 1 , Ana c 2 Act d 1 , and Car Act d 1 , respect Act d 1  from papPLCPs can cause occupational allergic reactions due to exposure to airborne particles, as reported, for instance, for papain ,31 or brIn this context, the aim of this study was the investigation of the frequency of sensitization by the measurement of IgE-positive results, and of allergic reactions to PLCPs that are present in raw plant foods and/or in products from the food industry. Therefore, this is an important subject of investigation because it falls into the food-safety topic. Here, the frequency of sensitization to five plant PLCPs  and, for comparison, two mite homologous enzymes, Der p 1 and Der f 1, was analysed by exploiting the FABER test, which allows for the detection of a specific IgE in the patients’ sera. A random Italian population reporting allergic symptoms was selected for this investigation. The study includes a comparative analysis of some structural features of the above-listed PLCP proteins. The results obtained in the analysed population show that compared to the mite homologs, the frequency of sensitization to the plant food proteases is lower and the clinical data suggest that the allergic reactions to the ingestion of plant food PLCPs might be rare.D. pteronyssinus and D. farinae , C. papaya, , A. comosus, , and F. carica . Act d 1 was purified from A. deliciosa, following an already reported procedure [The PLCPs analysed in this study are from rocedure .PLCPs are encoded by multigenic families expressing several protein isoforms. A multiple sequence alignment of all the available isoforms of cysteine proteases deriving from each organism under investigation would be very complex. Therefore, to make the analysis easier, a representative sequence for each organism/type of enzyme has been chosen and used for comparative investigation. As a general criterion of isoforms selection, curated sequences of the molecules really detected at the protein level have been chosen. Protein isoforms were registered in UniProt with a code having P as the first letter, followed by a number, where preferred, when available. In fact, this kind of code is generally a synonym of a reviewed protein sequence corresponding to a molecule isolated as a protein (rather than as a DNA sequence) and, very likely, it was present in high amounts in the natural source. Quite recently, the coding system of UniProt has been changed and, therefore, it is now more difficult to deduce some information from the accession number format. For instance, simply looking at the protein code, it is now not possible to understand if the sequence is gene-deduced or if the natural protein was isolated and characterized.www.uniprot.org, accessed on 26 May 2023). Thereby, a multiple sequence alignment was obtained with the program Clustal O . The output data also contained additional information, including the percentage of amino acid identity between the different protein sequences.Amino acid sequences representative of PLCPs of mites, kiwifruit, papaya, pineapple and fig were selected and aligned choosing “Align” on the menu of the UniProt website  is a multiplex in vitro serological test allowing for the detection of specific IgE antibodies produced by allergic subjects ,36. The The data here described were obtained from a set of biochips including, in addition to the standard 244 allergenic preparations, also allergens spotted for experimental purposes, such as the ficin protein (Fic c Ficin). Before the immobilization on the FABER biochip, ficin and the other proteins tested for experimental purposes were coupled to nanobeads following the same procedure applied to all the allergenic preparations.The IgE profile of 341 Italian patients tested with the FABER system was analysed. Their average age was 33.5 years and 186 (54.5%) patients were female. This was considered a random cohort, representing a general population of allergic subjects since no selection criteria were applied. A reliable clinical history was available for each one. The symptoms reported by patients, and other details including the use of medications, were recorded in a specific questionnaire, as described in a previous article . All invActually, the analysis of 14 sequences of Ana c 2 reveals the level of differences between the individual isoforms and suggests possible immunological and functional variability. The comparison shows that the only isoform lacking the Asn residue involved in the catalytic activity is the one with accession number P14518 . The AnaThe Ana c 2 isoform registered by WHO/IUIS as an allergen (Ana c 2.0101) is the molecule with UniProt accession number O23791, which is devoid of the N-glycosylation site found in the isoforms with accession numbers P14518, O23799, and O81084 . The amiThe allergic symptoms referred to by patients on the occasion of the first visit, during the collection of the anamnesis, are summarized in Symptoms like conjunctivitis, asthma, and urticaria were also reported with a frequency of around 30%. Rhinitis and conjunctivitis were mostly associated with patients complaining of symptoms in the spring. Lower frequency values were observed for symptoms like oral allergy syndrome (OAS), gastritis, enteritis, eczema, colitis, angioedema, and anaphylaxis due to other suspected allergenic sources where PLCP has not been reported yet. Kiwifruit ingestion caused OAS in one patient and OAS and urticaria in another one. One patient referred to fig pruning itch and two patients claimed OAS ingesting fig fruit. Twenty subjects (5.8%) referred to not eating fruits, including fig, kiwi, papaya, and pineapple. Among the 321 remaining patients, none claimed symptoms of ingesting papaya or pineapple.The sera (341) from patients reporting allergic symptoms towards any food or inhalant source were analysed with the FABER test ,37. The Despite sensitization to the cysteine proteases, no patients reported allergy symptoms to pineapple, papaya, and fig, whereas the three subjects reporting OAS against fig , followed by conjunctivitis and urticaria. In this context, FABER is the only diagnostic test that has been exploited for a comparative analysis of specific IgE towards plant fruit PLCPs from kiwifruit, papaya, pineapple, and fig, as well as the homologs Der p 1 and Der f 1 from mites. Out of 341 patients, 39% had IgE specific for at least one of the seven cysteine proteases under investigation, thus suggesting that the sensitization towards this protein family is not of negligible meaning. However, the results obtained for the individual allergens highlighted that the two mite homologs displayed the highest frequency of IgE-positive results (around 20%), with the patients sensitized to at least one of the two mite proteases corresponding to 24.3%. In line with the high sequence identity between Der p 1 and Der f 1, most of the patients who were IgE positive to a mite protease were cosensitized to the other one. Such findings are well known since their discovery ,70. It iIn contrast to Der p 1 and Der f 1, the food PLCPs did not show the same high correlation between IgE positivity and allergy reactions. For instance, the frequencies of IgE-positive results obtained for the two cysteine proteases from papaya fruit, Cari p 2, and Cari p Papain, and for the pineapple protein, Ana c 2, were 2.6%, 1.5%, and 12%, respectively, but none of the patients reported symptoms following the exposure to these foods. We cannot exclude that the higher value (12%) of IgE-positive results obtained for Ana c 2 could be affected by CCD-specific IgE that could be contained in some patients’ sera. Nevertheless, we observe IgE positivity not correlated with clinical allergy not only on Ana c 2 but also on Cari p 2, Cari p Papain, and Fic c Ficin.The frequency of IgE-positive results obtained by testing Act d 1, was lower (0.88%) compared to the homologs. However, only one of these patients reported allergy symptoms (OAS and urticaria) after the kiwifruit consumption. A second patient reported OAS after kiwifruit ingestion but the IgE negative tests to Act d 1 suggest that the allergy symptoms have to be attributed to other possible allergens contained in this food. In the case of Act d 1, we observe that the number of patients IgE positive to the allergen, and clinically allergic to kiwifruit, is low and does not allow for the drawing of meaningful conclusions.A non-negligible percentage of patients (8.5%) was IgE positive to the fig cysteine protease but none of them reported allergy symptoms following the exposure to this fruit. Actually, out of 341, three patients (0.88%) claimed allergy reaction to fig, two of them reporting OAS to the fruit ingestion and the third one reported an itchy reaction following skin contact to pruning but none of them was IgE positive to Fic c Ficin. Indeed, the literature reports describe that cysteine proteases can evoke itch in the absence of any allergic reaction ,72. TherIn summary, as reported for other allergens ,73, a stIn conclusion, at least in the investigated Italian population, sensitization to the mite cysteine proteases, Der p 1 and Der f 1 occurs with a higher frequency, compared with homologs from plant foods, such as kiwifruit, pineapple, papaya and fig. More importantly, the observed correlation between clinical allergy to mites and sensitization to Der p 1 and Der f 1 is very high, thus producing a high predictive value of allergy reactions by in vitro IgE tests. In contrast, the IgE-positive results obtained for the analysed plant food homologs do not correlate with allergy symptoms towards the protease source. In fact, despite the frequent exposure to PLCPs contained in raw fruits or included as additives in processed foods, the allergic reactions were generally absent in the sensitized patients analysed in this study. The only exception was represented by a patient with allergy reactions to kiwifruit and showing positive IgE binding to Act d 1. This is in line with the literature reports describing the reactions to some fruit cysteine proteases as allergy symptoms generally associated with occupational work. Therefore, as some of these cysteine proteases are known to cause allergy reactions in the exposed workers and to be able to sensitize the atopic subjects, it is likely that they might represent a risk of allergy in some populations. Nevertheless, this occurrence was not observed in the population examined in this study. Further investigations also performed with the inclusion of additional PLCPs on the biochip of diagnostic systems will make human biomonitoring studies possible.


In this study, a 200 μL incubation system was used to study the inhibitory effect of the antitumor drug napabucasin on arbidol in vitro, with IC50 values of 2.25, 3.91, and 67.79 μM in rat liver microsomes (RLMs), human liver microsomes (HLMs), and CYP3A4.1, respectively. In addition, we found that the mechanism of inhibition was non-competitive inhibition in RLM and mixed inhibition in HLM. In pharmacokinetic experiments, it was observed that after gavage administration of 48 mg/kg napabucasin and 20 mg/kg arbidol, napabucasin inhibited the metabolism of arbidol in vivo and significantly changed the pharmacokinetic parameters of arbidol, such as AUC(0-t) and AUC(0-∞), in rats. We also found that napabucasin increased the AUC(0-t) and AUC(0-∞) of M6-1, the main metabolite of arbidol. This study provides a reference for the combined use of napabucasin and arbidol in clinical practice.As a broad-spectrum antiviral, and especially as a popular drug for treating coronavirus disease 2019 (COVID‐19) today, arbidol often involves drug–drug interactions (DDI) when treating critical patients. This study established a rapid and effective ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method to detect arbidol and its metabolite arbidol sulfoxide (M6-1) levels  In 1993, arbidol was introduced as a broad-spectrum antiviral drug to prevent and treat influenza infection . Arbidolmax of arbidol was increased and the AUC(0-∞) of arbidol was significantly increased from 705.6 to 1250.3 ng/mL*h  and arbidol will be a risk factor for liver injury in patients with non-critical COVID-19 . Studies ng/mL*h . In addi ng/mL*h , the levIn cancer treatment, napabucasin is a therapy that targets signal transducer and activator of transcription 3 (STAT3) pathways . Severalin vitro and in vivo.Drugs are often used in combination during the treatment of cancer, as cancer patients were also found to be affected with COVID-19 . HoweverArbidol and its metabolite, M6-1, were obtained from Shanghai Canspec Scientific Instruments Co., Ltd. . Lopinavir, used as the internal standard (IS), and napabucasin were also purchased from Shanghai Canspec Scientific Instruments Co., Ltd. . Analytical instruments were filled with acetonitrile and methanol that were purchased from Merck . Human liver microsomes (HLMs) and CYP3A4.1 were obtained from iPhase Pharmaceutical Services Co., Ltd. . Reduced nicotinamide adenine dinucleotide phosphate (NADPH) was obtained from Roche Pharmaceutical Ltd. . A Milli-Q Ultrapure Water System  was used to produce purified water.Quantitative analysis was performed using a combination of the Waters Xevo TQ-S triple quadrupole tandem mass spectrometer and a Waters ACQUITY UPLC I-Class system . The instrument was equipped with an automatic sample injection program to achieve fully automatic sample filling and accurate detection. The ACQUITY BEH C18 column  used for accurate separation of the analytes in the sample at 40°C with acetonitrile (solvent A) and 0.1% formic acid (solvent B) as mobile phases. The gradient elution program was conducted at a flow rate of 0.4 mL/min as follows: 0–0.5 min (10% A+ 90% B), 0.5–1.0 min (10% A increase to 90% A), 1.0–1.4 min (maintain 90% A+ 10% B), 1.4–1.5 min (90% A decrease to 10% A), and 1.5–2.0 min (10% A+ 90% B). Triple quadrupole mass spectrometers were equipped with electrospray ionization (ESI) sources, and multiple reaction monitoring (MRM) modes were used for quantification. Depending on the ion, we selected different ion monitoring voltage conditions, and the results are shown in Rat livers were weighed and homogenized with cold 0.01 mM phosphate-buffered saline  containing 0.25 mM sucrose. The homogenates were centrifuged at 11,000 rpm at 4°C for 15 min, and the supernatants were centrifuged repeatedly. Then, the supernatants were transferred to new tubes, centrifuged at 75,600 x g for 2 h, the supernatants were discarded, and three or four times the volume of cold 0.01 mM phosphate-buffered saline was added to the precipitate for homogenization. Finally, the protein concentrations were determined using the Pierce™ BCA Protein Assay Kit (Thermo Scientific) .m (Michaelis constant) in RLM. In HLM, a series of concentrations  of arbidol were used to determine Km. In CYP3A4.1, the following concentrations of arbidol were used to determine Km: 1, 10, 20, 50, 150, 200, 250 μM. Napabucasin was established at concentrations 0.01, 0.1, 1, 10, 25, 50, and 100 μM to determine its IC50  versus arbidol . In addition, the changes in Michaelis–Menten curves in the presence of napabucasin were investigated in RLM and HLM. To study the type of inhibition mechanism, we used the Lineweaver–Burk plot analysis and inhibition constant (Ki and αKi) calculation, where drug concentrations were set as follows: 1.17, 2.33, 4.66, and 9.32 μM for arbidol and 0, 1.13, 2.25, and 4.50 μM for napabucasin in RLM. In HLM, the concentrations of arbidol were 0.66, 1.32, 2.63, and 5.26 μM, and those of napabucasin were 0, 0.98, 1.95, and 3.90 μM. The components in the incubation system were pre-incubated at 37°C for 5 min. After adding NADPH, the reaction was initiated and incubated at 37°C for 30 min. After the stop reaction at −80°C, 400 μL of acetonitrile and 20 μL of IS (lopinavir 200 ng/mL) were added for post-treatment, vortexed for 2 min, and centrifuged at 13,000 rpm for 10 min. Finally, the supernatant was used for quantitative analysis using UPLC-MS/MS.The 200 μL incubation system was as follows: 0.3 mg/mL RLM (0.3 mg/mL HLM), 1 mM NADPH, pH 7.4 phosphate buffer, and arbidol. Arbidol was used in a range of concentrations  to determine KSprague–Dawley rats  were supplied by The First Affiliated Hospital of Wenzhou Medical University and used for the pharmacokinetic experiments. Except for fasting 12 h before the start of pharmacokinetic experiments, rats were fed on a standard rodent diet and kept in a 12-h light–dark cycle environment at 20°C–26°C and 55 ± 15% relative humidity.Rats were randomly divided into two groups of five each. Arbidol and napabucasin were soluble in 0.5% carboxymethylcellulose sodium solution (CMC-Na). The treatment group was given 48 m/kg napabucasin by gavage, and the control group was given 0.5% CMC-Na solution of the same volume. Thirty minutes after the treatment, each rat was given a single administration of 20 mg/kg of arbidol. Blood samples were obtained from the caudal veins at different times . A volume of 10 μL of IS (200 ng/mL) and 300 μL of acetonitrile was added to 100 μL of plasma and mixed in the Eppendorf tube. The UPLC-MS/MS analysis of the supernatant was conducted after centrifuging the mixture at 13,000 rpm for 10 min.50 values and enzyme kinetic parameters were carried out using GraphPad Prism 6.0 . The key pharmacokinetic parameters were analyzed using DAS software . Means ± standard deviations (SD) were calculated for each experiment.Calculations of ICr2 > 0.99. Both arbidol and M6-1 had acceptable precision and accuracy with a low limit of quantitation (LLOQ) of 1.0 ng/mL.The methodologies were compliant with the standards of the EMA and FDA for bioanalytical methods . In thism of arbidol was 4.66 μM, the IC50 of napabucasin for arbidol was 2.25 μM, and the metabolic rate of arbidol was decreased to 3.73% in the presence of 100 μM napabucasin. In HLM, the Km of arbidol was 2.63 μM and the IC50 of napabucasin for arbidol was 3.91 μM  were 0.77 and 0.98 μM, respectively (α = 0.54) were 1.59 and 0.86 μM, respectively  of arbidol in rats in the treatment group was increased by 1.85-fold, and the AUC(0-∞) was increased by 1.59-fold. In addition, the parameters CLz/F were reduced by 58.0% ((0-t) of M6-1 in rats from the treatment group was increased by 78.2%, and AUC(0-∞) was increased by 93.1%. Interestingly, the parameters CLz/F were reduced by 47.1%  and the treatment group (arbidol with napabucasin) is shown in by 58.0% , which iby 47.1% . Based oArbidol is a popular drug for the treatment of COVID-19, and its pharmacokinetic metabolism in the body is gradually attracting attention . In the 50 < 10 μM, it was considered medium inhibitory efficiency . To prevent adverse reactions, it is recommended to pay attention to DDI when using chemotherapy drugs with arbidol to treat diseases in the clinic. Arbidol inhibition studies with napabucasin showed that there were differences between species in vitro. The type of inhibition mechanism was non-competitive inhibition in RLM, while the inhibition type was mixed inhibition in HLM. In addition, in HLM, Ki and αKi (α = 0.54) were 1.59 and 0.86 μM, respectively. However, the mechanism in CYP3A4.1 was not investigated due to its excessive IC50.The chromatogram analysis results of UPLC-MS/MS showed that there was no interference between arbidol, M6-1, and IS during the detection process. When 1 < ICficiency . The metmax and AUC(0-∞) of both drugs  and AUC(0-∞)  . Under tin vivo and in vitro. The differences in inhibitory effects in RLM and HLM may come from differences between species, as the amount of CYP3A4 expressed was not the same between species. Previous studies reported that differences in sexes can be observed in arbidol metabolism, but unfortunately, this experiment did not compare the metabolic differences of napabucasin and arbidol between rat sexes. A future study in clinics should investigate the effect of napabucasin on arbidol metabolism in humans and whether gender affects the interaction between the two drugs.The aforementioned results showed that napabucasin inhibited arbidol metabolism in vitro, except for CYP3A4.1, with weak inhibition. Its inhibition mechanism was non-competitive in RLM and mixed inhibition (competitive and non-competitive) in HLM. Moreover, in rat pharmacokinetic studies, napabucasin inhibited the metabolism of arbidol and its main metabolite M6-1 in a subtle way. In future studies, more attention should be paid to the combination of these two drugs, and the DDI in humans needs to be further explored.The metabolism of arbidol in RLM and HLM was moderately inhibited by napabucasin


While we observe significant trends in SST, sea ice, and chlorophyll-a/primary productivity throughout the year, the most significant and synoptic trends for the DBO sites have been those during late summer and autumn . Those DBO sites where significant increases in annual primary productivity over the 2003–2020 time period have been observed include DBO1 in the Bering Sea (37.7 g C/m2/year/decade), DBO3 in the Chukchi Sea (48.0 g C/m2/year/decade), and DBO8 in the Beaufort Sea (38.8 g C/m2/year/decade). The length of the open water season explains the variance of annual primary productivity most strongly for sites DBO3 (74%), DBO4 in the Chukchi Sea (79%), and DBO6 in the Beaufort Sea (78%), with DBO3 influenced most strongly with each day of additional increased open water (3.8 g C/m2/year per day). These synoptic satellite-based observations across the suite of DBO sites will provide the legacy groundwork necessary to track additional and inevitable future physical and biological change across the region in response to ongoing climate warming.Massive declines in sea ice cover and widespread warming seawaters across the Pacific Arctic region over the past several decades have resulted in profound shifts in marine ecosystems that have cascaded throughout all trophic levels. The Distributed Biological Observatory (DBO) provides sampling infrastructure for a latitudinal gradient of biological “hotspot” regions across the Pacific Arctic region, with eight sites spanning the northern Bering, Chukchi, and Beaufort Seas. The purpose of this study is two-fold: (a) to provide an assessment of satellite-based environmental variables for the eight DBO sites (including sea surface temperature (SST), sea ice concentration, annual sea ice persistence and the timing of sea ice breakup/formation, chlorophyll- Arctic sea ice cover is undergoing extensive changes. There has been a pronounced decrease in summer sea ice extent , an overThe Pacific Arctic sector is also among the most biologically productive marine ecosystems in the world –14 and aa concentrations, primary productivity, and photosynthetically available radiation (PAR)) as well as trends across the 2003–2020 time period; and (b) to assess the impact of sea ice presence in winter and the length of open water in spring, summer, and autumn on primary productivity across the region and for the eight DBO sites in particular.In 2009, it was proposed to develop a Distributed Biological Observatory (DBO) across the Pacific Arctic sector to monitor shifting marine ecosystems in response to recent dramatic sea ice declines and seawater warming in the region . As a rehttps://www.ncei.noaa.gov/products/optimum-interpolation-sst. These data have been validated and compared with other data sources such as those from Aqua-Moderate Resolution Imaging Spectroradiometer (MODIS) )a concentration, primary productivity, and PAR (as described above) were compiled over the years 2003–2020, which is the time period when Aqua-MODIS data are available . Annual mean values of primary productivity (March–September only), the timing of sea ice breakup and formation, and sea ice persistence were additionally calculated across the region for the years 2003–2020. Next, spatial trends using the Theil-Sen median slope estimator [a, primary productivity, and PAR data. The Theil-Sen trend uses a robust non-parametric trend operator that is particularly well suited for assessing the rate of change in noisy and/or short time series [p<0.1) was established using the non-parametric Mann-Kendall test for monotonic trend [Our statistical analyses were applied to all time series datasets consistently. First, monthly mean values of SST, sea ice concentration, chlorophyll-stimator  were cale series , which iic trend ,74. For ic trend ). Lastlya concentration, and primary productivity) show a distinct latitudinal gradient across all DBO sites as well as strong seasonality within each individual DBO site  of environmental variables investigated , falling in June-July, then increasing again in August and September . Chlorophyll-a concentrations at sites DBO4-DBO8 show seasonal variability, however over a shorter season given the available ice-free data at these sites. In particular, DBO4 exhibits its highest concentrations during May-June, lowest concentrations in June-July, then a slight uptick in September. DBO5 and DBO7 show similar chlorophyll-a patterns with increases from May-July, falling in August, then increasing slightly in September. DBO6 exhibits only four months of sea ice-free chlorophyll-a data, increasing from June-July, decreasing in August, then increasing slightly in September. DBO8 is the only site that does not exhibit a mid-summer decrease in chlorophyll-a concentrations, continuously increasing over the ice-free months at that location, from May-September. Corresponding rates of primary production (a concentrations) is much less apparent for the DBO sites in general. This is likely at least in part due to cooling SST in September and October not contributing to as high primary productivity compared to earlier in the season (June-August). Similarly, while chlorophyll-a concentrations tend to peak early in the season (within weeks of sea ice breakup), primary productivity tends to peak later in the season (despite lower chlorophyll-a concentrations) perhaps because of the contributions of warmer mid-summer SST to these rates and/or varying photosynthetic rates among phytoplankton groups [2/year) and DBO3 (214.5 g C/m2/year); the lowest values at DBO4 (88.3 g C/m2/year), DBO5 (74.5 g C/m2/year), and DBO6 (68.7 g C/m2/year); and middle-range values at DBO1 (144.5 g C/m2/year), DBO7 (108.9 g C/m2/year), and DBO8 (114.6 g C/m2/year)  . Clear sa, primary productivity, and PAR. As such, the Theil-Sen formula is used to estimate the median trends for all variables for each month, and annually in the case of sea ice persistence/breakup/formation and annual primary productivity. For SST, warming trends are pervasive across the region for all months  variability e.g., . During a  and the southern Chukchi Sea during July, August, and September. Fewer statistically significant trends occur during October, but data are spatially limited during this month owing to the onset of polar night. Compiling data into trends of annual primary productivity also helps to identify regions of greatest importance of change , August (DBO3 = 0.67 mg/m3/decade), September (DBO3 = 1.2 mg/m3/decade and DBO6 = 0.51 mg/m3/decade). Significant negative trends occur for DBO2 during June (-0.73 mg/m3/decade). For primary productivity, significant positive trends occur for June (DBO1 = 167 mg/m2/day/decade), August (DBO3 = 243 mg/m2/day/decade and DBO4 = 167 mg/m2/day/decade), and September . Significant negative trends in primary productivity occur for DBO2 during June (-354 mg/m2/day/decade). Trends in annual primary productivity are significant at DBO1 (37.7 g C/m2/year/decade), DBO3 (48.0 g C/m2/year/decade), and DBO8 (31.2 g C/m2/year/decade). Lastly for sea ice events, breakup trends are significant at DBO1 (-21.9 days/decade) and DBO3 (-8.4 days/decade). More trends in the timing of seasonal sea ice formation are significant, including at DBO2 (12.9 days/decade), DBO3 (12.6 days/decade), DBO4 (10.5 days/decade), DBO7 (10.5 days/decade), and DBO8 (10.6 days/decade). Trends in annual sea ice persistence are significant at DBO1 (-32.3 days/decade), DBO3 (-16.6 days/decade), DBO4 (-29.3 days/decade), DBO5 (-15.6 days/decade), DBO7 (-30.4 days/decade), and DBO8 (-19.8 days/decade).While we can summarize the overall trends for each of the eight DBO sites, it is important to note that the spatial patterns in statistically significant trends are such that the DBO sites are not necessarily representative of the regions of greatest change in SST, sea ice, chlorophyll-BO sites . While t2/year) and DBO7 (178.5 g C/m2/year), and the smallest interannual ranges are at DBO5 (92.3 g C/m2/year) and DBO6 (124.9 g C/m2/year). As a result of the linear regressions, we see that the highest R2 values indicate that ~79% of the variance in mean annual primary productivity can be explained by the length of the open water season at DBO4, 78% for DBO6, and 74% for DBO3. DBO sites where open water explained the least of the variance in annual primary productivity include DBO7 (36%) and DBO2 (47%). In addition, the steepest slope of the primary productivity-open water relationship is found at DBO3 (3.8 g C/m2/year per day), meaning that for every day gained of the annual open water season (over the March-September period), primary productivity increases 3.8 g C/m2/year. The spatial representation of slope and R2 also provides important context for this primary productivity-open water relationship across the Pacific Arctic region (2/year per day) across the study region in the southern Chukchi Sea just north of Bering Strait in the vicinity of DBO3  is strong across the region  seem to congregate along the sea ice edge in the northern Bering Sea as well as in the western Bering Strait. This western Bering Strait region of low R2 values (encompassing portions of both DBO2 and DBO3) geographically coincides with some of the negative primary productivity-open water slopes  for both the individual eight DBO sites  and spatc region . We obse of DBO3 . All sloe region , with mor slopes  as well lopes  for all eight DBO sites across the Pacific Arctic region. While the DBO sites are often described as distributed across a latitudinal (or climate) gradient, there are exceptions to this general notion. In particular, although there is an expected clear cooling pattern with longer seasonal sea ice cover moving from DBO1 in the south to DBO6 in the north, DBO7 and DBO8 show warmer temperatures and less sea ice cover (compared to DBO6), which is likely associated with the warming effects of the Beaufort Gyre [a and primary productivity across the DBO sites, however these parameters have more complex variability because they are also affected by other factors such as nutrient availability, length of daylight, and grazing. In general, DBO2 and DBO3 have markedly higher mean annual primary production than any other DBO site , which is in large part sustained by nutrient-rich waters along the western portion of the Bering Strait region [a concentrations nor primary production returns to the levels observed during spring  . However, this does not necessarily mean that important changes have not also occurred earlier in the season during spring. In fact, warming SSTs  does not seem associated with cooling trends of SST . Sea ice variability is controlled by multiple drivers, including cooling or warming air temperatures, changes in atmospheric circulation and ice motion, shifts in cloud cover, and advected ocean heat [As highlighted previously, the most significant and synoptic trends for the DBO sites have been those during late summer and autumn , which ean heat ,79. Howeean heat .2/year) for every day of open water added to the season. This may indicate that nutrients are sufficiently available at these sites and that low light availability owing to sea ice presence still remains a primary limitation of annual production. However, with the expected continuation of sea ice decline and a lengthening open water season throughout the Pacific Arctic region , the primary limitations on production may continue to shift. While patterns in sea ice and primary productivity for 2021 and 2022 have been spatially heterogenous across the Pacific Arctic region [While an increased supply of nutrients has been shown to be a dominant driver of increased primary productivity in recent years across the Arctic Ocean , our stuc region ,90, neitc region ,9). NeveS1 File(PDF)Click here for additional data file.


A unique feature of IF1 is condition-dependent inhibition; although IF1 inhibits ATP hydrolysis by F1, IF1 inhibition is relieved under ATP synthesis conditions. To elucidate this condition-dependent inhibition mechanism, we have performed single-molecule manipulation experiments on IF1-inhibited bovine mitochondrial F1 (bMF1). The results show that IF1-inhibited F1 is efficiently activated only when F1 is rotated in the clockwise (ATP synthesis) direction, but not in the counterclockwise direction. The observed rotational-direction-dependent activation explains the condition-dependent mechanism of IF1 inhibition. Investigation of mutant IF1 with N-terminal truncations shows that the interaction with the γ subunit at the N-terminal regions is crucial for rotational-direction-dependent ejection, and the middle long helix is responsible for the inhibition of F1.IF 1 is a natural inhibitor of mitochondrial FoF1-ATP synthase, which blocks catalysis and rotation of the F1 motor. Here, the authors show the rotational-direction-dependence of activation from IF1 inhibition, with IF1 being readily dissociated when F1 rotates to the clockwise direction.IF FoF1 is composed of two rotary motor proteins: Fo and F15. Fo is a membrane-embedded protein complex forming the proton translocation pathway. When FoF1 synthesizes ATP, Fo transports protons from the outside to the inside of the membrane, accompanied by the clockwise rotation of the rotor complex inside FoF1 when viewed from the outside of the membrane. F1 is a water-soluble portion of FoF1 that contains catalytic centers for ATP synthesis. Proton translocation by Fo and ATP synthesis/hydrolysis reactions in F1 are tightly coupled through the rotation of the rotor complex. When pmf is sufficiently high, Fo forcibly rotates F1, powered by proton translocation down pmf, resulting in ATP synthesis via F1. When pmf is insufficient, F1 reverses the rotation and hydrolyzes ATP, which induces Fo to pump protons to generate pmf 6.F1 is an ATPase that hydrolyzes ATP to rotate its rotor part counterclockwise, when viewed from the membrane side, against the surrounding stator α3β3-ring7, where the central rotor γ subunit is accommodated in the central cavity12. F1 possesses three catalytic sites for ATP hydrolysis/synthesis, each at the interface between the α and β subunits. Amino acid residues critical for catalysis are mainly located in the β subunit. The “ground-state” crystal structure of bMF113 revealed that the three β subunits have different conformations and nucleotide-bound states; β with nucleotide analog (βTP), β with ADP (βDP), and β with none (βE). The βTP and βDP adopt a closed form with their C-terminal domain swinging towards the γ subunit, whereas βE adopts an open conformation14. The rotary dynamics of F1 have been investigated extensively using single-molecule studies19. The reaction scheme of rotary catalysis in bMF120 21. Several experiments have shown that most of the crystal structures of bMF1 represent catalytic dwell states24.F120 Fig.  was propoF1 play the primary role in ATP synthesis in vivo, although proton- or sodium-pumping activity driven by ATP hydrolysis is dominant in some species. In general, ATP hydrolysis activity of F1 can result in futile ATP consumption. Therefore, several types of regulatory systems can suppress or block the ATP hydrolysis activity of F130. The most common mechanism universally found in bacterial and mammalian F1 is ADP inhibition in which F1 spontaneously lapses into a resting state during rotation33. Mitochondrial F1s have a unique inhibitor protein, termed the ATPase inhibitory factor 1 (IF1)34. It is found in eukaryotic cells and is highly conserved particularly among mammalian cells25. Unlike the ε subunit38 that some of the bacterial F1s employ for inhibition, IF1 is not a built-in inhibitor, but a dissociative one that associates with F1 when pH decreases to acidic state or F1 is isolated before association with Fo to form the whole complex of FoF16. The most remarkable feature of IF1 is its condition-dependent manner of inhibition: IF1 inhibits ATP hydrolysis activity of F1 almost completely under ATP hydrolysis conditions, whereas, in ATP synthesis conditions, IF1 dissociates from F1 and does not interfere with ATP synthesis reactions after dissociation39. Notably, a few recent studies claim that IF1 also suppresses the rate of ATP synthesis when IF1 is overexpressed in cells41.Most F1 and bMF1 have been investigated in structural studies44. Full-length bovine mitochondrial IF1 forms a homodimer complex via an antiparallel coiled coil45, which associates two molecules of the F1-ATPase with each N-terminal region, whereas the C-terminal region of IF1 forms a coiled-coil structure for dimerization42. Deletion of the C-terminal residues 61–84 produces a stable monomeric form of IF1, which achieves full inhibition capacity irrespective of pH change46. This simple platform of IF1 is often used in biochemical48 and structural studies44, including this study. The crystal structure of the bMF1-IF1 complex44 showed that the long α-helical structure in the middle section of IF1 was bound to the interface of the αβDP pair, whereas the short helix of the N-terminus was in contact with the γ subunit. The N-terminal short helix was linked to the middle-long helix via a distinct kink 3, has been resolved43, where each αβ interface was bound with IF1. Because the structures of the αβ interface are different, IF1’s show different conformational states. The structure of IF1 on αβDP is consistent with that of the 1:1 bMF1-IF1 complex, whereas the N-terminal short helix of IF1 is not resolved on αβTP. The structure of IF1 on αβE shows the second half of a long helix, and the remaining was unsolved. Based on these observations, the progressive folding of IF1 coupled with γ rotation has been proposed. Progressive folding process has also been suggested in biochemical studies49, although the progressive processes are simplified as a two-step process: the initial binding process and the subsequent isomerization process.The atomic details of the interaction between IFink Fig. . The cry51, studies on IF1 under synthetic conditions are less advanced, despite its importance for the understanding of the condition-dependent inhibition mechanism of IF1. IF1-inhibited state of F1-ATPase is known to be so stable that F1 is unable to eject IF1 by thermal agitation alone48. A typical condition for the unlocking from IF1 inhibition is to charge sufficiently high pmf to membrane vesicles containing IF1-inhibited FoF157. However, the principal mechanism for IF1 ejection from the catalytic site of F1, followed by the recovery of catalysis in F1 , remains elusive. Many fundamental questions are unsolved such as ‘Does reversible rotation lead to activation from IF1 inhibition?’, ‘Are there factors to enhance the activation?’, and ‘Which interactions among IF1 and F1 are responsible for IF1 inhibition and for condition-dependent inhibition?’.Compared to studies under hydrolysis conditions1 from the inhibition complex using magnetic tweezers, which enable control of the angular orientation of the rotor during single-molecule rotation assays for bMF1  attached to the γ subunit as a rotation probe and recorded at 30 frames per second (fps). During rotation, bMF1 showed a transient pause in the absence of IF120. This was attributed to ADP inhibition. Supplementary Fig. 1 inhibition, monomeric bovine IF1 (Δ61–84) was used, as described in previous studies48.The rotation of 1-inhibited bMF1 molecules was as follows 20 and sodium azide  to prevent possible rebinding of IF1. However, the molecules did not show activation in most cases , and then, IF1-inhibited bMF1 molecules were forcibly rotated in the clockwise or counterclockwise direction for 360°. As shown in Fig. 1-inhibited bMF1 was rotated in the clockwise direction in the presence of ADP and Pi; the probability of activation was about 60%. This is in contrast to the reactivation probability for the clockwise manipulation in ADP/Pi-free solution (10%). An evident rotational-direction-dependent manner was observed; the counterclockwise rotation failed the activation. These observations indicate that both directional manipulation and the presence of substrates for ATP synthesis are requisite for the efficient activation of the IF1-inhibited state. To investigate the effect of individual substrates on activation, we performed the same manipulation experiment in the presence of ADP or Pi, respectively , whereas the activation achieved only 4% in the presence of ADP. Notably, once activated, bMF1 molecules did not show long pauses that are attributable to IF1 inhibition in IF1-free solution. This observation indicates that activation is accompanied by dissociation of IF1 from F1.Next, we tested the effect of ADP and P1 inhibition, the inhibited bMF1 molecules were rotated to stall at the target angle for the programmed period of 0.5–5.0 s with the magnetic tweezers. After the set time had elapsed, the magnetic tweezers were turned off to release the F1 molecule. The released molecule showed two types of behaviors , indicating that re-inactivation was due to the rebinding of IF1 from solution. We repeated the manipulation of each molecule to confirm reproducibility. After manipulation, a few molecules occasionally exhibited unusual behaviors, such as random tethered Brownian motion, and nonspecific binding to or detachment from the coverslip. These data were omitted from the analyses. A detailed description of the analysis of molecules in this experiment is presented in Supplementary Note and Supplementary Fig. We determined the activation probability as a function of the rotation angle by performing a “stall-and-release” experiment. The experimental procedure was as follows Fig. ; after cors Fig. : startini. Fig 1-inhibited state. In principle, activation was rarely observed in the counterclockwise manipulation, which is consistent with the experimental results for forcible 360° rotation manipulation , unstructured region (8–13), the short helix region (14–18), glycine loop (19–20), and the long helix region (21–60) , IF1(Δ1–12), IF1(Δ1–19), and IF1(Δ1–22)  and IF1(Δ1–12) showed similar inhibitory effects to IF1(WT); upon association with bMF1, the mutants of IF1 completely inhibited ATPase activity. In contrast, IF1(Δ1–19) and IF1(Δ1–22) showed a reduced inhibitory effect. At concentrations lower than 0.5 μM, IF1(Δ1–19) did not completely inhibit ATPase activity, suggesting a reversible association/dissociation. IF1(Δ1–22) showed a lower inhibitory effect with a peculiar kinetic behavior. Upon addition of IF1(Δ1–22) into the assay mixture, ATPase activity decreased, followed by slow recovery. Although the mechanisms underlying such complex behavior are unclear, these biochemical measurements showed that the inhibitory effect of IF1(Δ1–19) and IF1(Δ1–22) was less than that of IF1(Δ1–7) and IF1(Δ1–12).With an aim to investigate which structural elements of IFnts Fig. . IF1 con60) Fig. a, b. TheF22 Fig. 43. We pr22) Fig. . The inhd region –13, the ne loop 1–20, and bMF1 in the presence of truncated mutants of IF1. Similar to the biochemical experiments, we observed that IF1(Δ1–7) and IF1(Δ1–12) showed nearly the same properties as those of IF1(WT)  and IF1(Δ1–22), bMF1 molecules did not completely stop the rotation, but the molecules showed frequent transitions between rotation and intervening pauses  or IF1(Δ1–22) was 250 s or 60 s, respectively. These observations show that the short helix and N-terminus of the long helix of IF1 have critical roles in the stabilization of IF1 inhibition.We analyzed the rotation of 1’s. Whereas the activation experiments for IF1(Δ1–7) and IF1(Δ1–12) were conducted in IF1-free solutions, those were done in the presence of IF1 for IF1(Δ1–19) and IF1(Δ1–22). This is because these mutants can dissociate from F1 during buffer exchange, hampering the activation experiment. Fig 1(Δ1–7) and IF1(Δ1–12) showed similar trends to that of IF1(WT) in both directions; i.e., no activation after counterclockwise rotation, whereas clockwise rotation induced activation with a significant probability of 80%, which is higher than that for IF1(WT) at 58%. These results indicate that the unstructured N-terminal region from positions 1 to 13 of IF1 is not responsible for the principal mechanism of IF1 inhibition, although the region contributes to the structural stabilization of the bMF1-IF1 complex. Unlike the truncation of the unstructured N-terminal region, the truncation of the short helix and the subsequent N-terminal region of the long helix had the distinctive impact on rotation-direction-dependent activation. For the mutants IF1(Δ1–19) and IF1(Δ1–22), the activation probabilities after clockwise or counterclockwise rotation were both higher than those for IF1(WT), IF1(Δ1–7), and IF1(Δ1–12). In particular, a significant fraction of events showed activation after counterclockwise rotation: 13% for IF1(Δ1–19) and 80% for IF1(Δ1–22). The activation probability after clockwise rotation was also higher than that for IF1(WT), IF1(Δ1–7), and IF1(Δ1–12), and was close to 100%. These results suggest that IF1 readily dissociates from bMF1 without the short helix and the N-terminal tip of the long helix, as suggested by biochemical data. Importantly, IF1(Δ1–22) almost loses the rotation-direction-dependent feature, and the activation probabilities after clockwise or counterclockwise rotation are more than 80%. Considering that IF1 shows direct contact with the γ subunit in the truncated regions, these results suggest that the rotation-direction-dependent dissociation mechanism is based on contact with the γ subunit. One may consider that IF1(Δ1–22) dissociates from bMF1 not by forcible rotation, but by its nature of spontaneous dissociation. However, the manipulation time is 2 s which is too short for spontaneous dissociation of IF1(Δ1-22); the probability of spontaneous dissociation within 2 s is only 3% when estimated from the mean time constant of spontaneous activation, 60 s (see Supplementary Note). Thus, the observed activation with IF1(Δ1–22) results due to forcible rotation.Rotation-direction-dependent activation was investigated with the mutant IF1 inhibition by forcible rotation with magnetic tweezers. Activation is accompanied by the ejection of bound IF1 from bMF1. This is supported by the following observations: re-inactivation by IF1 was observed only when the solution contained IF1. In the absence of IF1, the activated molecules did not show long pauses attributable to IF1 inhibition. Second, the mean rotation time before re-inactivation was in good agreement with that for IF1 inhibition  direction. The activation probability was significantly enhanced in the presence of Pi in the solution by a factor of 15, although ADP also had an impact on the activation by a factor of 2 or more , the probability of activation reached 100% when stalled at –320° for 5 s. This is sufficiently high to explain the pmf-induced activation of the IF1-inhibited FoF157. Thus, the principal mechanism for the pmf-induced activation of IF1-inhibited FoF1 is the ejection of IF1 from F1 by forcible rotation powered by pmf-driven Fo motor in the FoF1 complex. The requirement of ADP and Pi for efficient activation indicates that forcible clockwise rotation should be coupled with the ATP synthesis reaction for IF1 ejection. As the presence of substrates enhances the cooperative nature of F1, IF1 may likely be ejected through a concerted conformational transition of the α and β subunits coupled with γ subunit rotation.In this study, we observed the activation from IF1-inhibited bMF1 was rotated over –200°  in the scheme. These results suggest that IF1 is ejected through the state transitions of the β subunit from the +200° state to the 0° state or –40° 3 structure, which suggests the progressive folding of IF1 coupled with the conformational transition of the β subunit from βE to βDP via βTP.We observed a significant increase in the probability of activation when the IF00° Fig. . The cry40° Fig. . This st1 inhibition is important to discuss under what conditions ATP synthase is activated in the cell. Although this study does not measure the torque of magnetic tweezers applied to F1 molecules, rough estimation is possible based on several assumptions. When bMF1 generates torque of 40 pN·nm like other F1s, the minimum value for proton motive force as voltage to reverse F1 is 196 mV, considering the proton stoichiometry of 8 protons/turn. In addition, the minimum energy for IF1 ejection is assumed to be 140 pN·nm (84 kJ/mol).Estimating the energy required for activation from IF1. This can be viewed as an inevitable property for IF1 to avoid being Maxwell’s demon that violates the second law of thermodynamics by allowing only one direction of motion in a microscopic system. If IF1 can dissociate from F1 without coupling with clockwise rotation, while IF1 association is tightly coupled with counterclockwise rotation, the second law of thermodynamics will be violated as Maxwell’s demon. This is evident when one considers the rotary motion of FoF1 under conditions where the torques of F1 and Fo are balanced. In IF1-free conditions, the rotor complex should show non-biased Brownian motion in both directions: clockwise and counterclockwise, in the FoF1 complex. Even though the rotor can cause occasional rotary steps in both directions, the mean rotational displacement does not increase. In contrast, in the presence of IF1, F1 should make a +200° rotation coupled upon the association of IF1. This is because ‘the tight coupling of IF1 association and the rotation’ means that IF1 association induces +200° rotation. Although the pause by IF1 inhibition should be long, IF1 should eventually dissociates from F1. Along the above assumption, IF1 is dissociated without biasing rotation. Thus, each cycle of association and dissociation of IF1 should cause the rotation by +200° in hydrolysis direction. After multiple events of IF1 association and dissociation, F1 undergoes unidirectionally biased rotation in hydrolysis direction. Thus, the assumption of the asymmetric coupling model should violate the second law of thermodynamics, and the reversibility of IF1 association/dissociation seems inevitable. For the same reason, the requirement for ADP and Pi for IF1 dissociation is also inevitable, considering that IF1 association is coupled with ATP hydrolysis.The above discussion indicates the reversible association/dissociation processes of IFoF1. In addition to IF1, several types of inhibitory mechanisms can block the ATP hydrolysis. The ζ subunit of α-proteobacteria inhibits hydrolysis by binding to the interface of the αβDP pair in a manner similar to IF163. Mycobacteria have a specific insertion sequence at the C-terminal of the α subunit called the α-extension loop65. Recent cryo-EM analysis showed that the α-extension loop binds to a specific loop on the γ subunit, which is considered to block rotation in a counterclockwise direction66. When these inhibitory processes are coupled with the rotation of the γ subunit, the dissociation of the ζ subunit or the α-extension loop has to be coupled with rotation in the opposite direction, according to the above contention.This consideration would be applicable to other regulatory systems of F1 kinetics at the physiological temperature of bovine mitochondria  is essentially the same as that at room temperature, the biochemical assay of IF1(WT), IF1(Δ1-19) and IF1(Δ1-22) at 37 °C was performed  showed complete inhibition with almost no residual activity, whereas IF1(Δ1-19) and IF1(Δ1-22) did not completely inhibit ATPase activity. These results suggest that the principle of IF1 inhibition and dissociation does not differ between room temperature to physiological temperature.All the experiments described in the main text were performed at room temperature; 23 ± 2 °C for single-molecule experiments and 25 °C for solution experiments. In order to confirm that IFbMF1-IF1 complex showed that IF1 has interactions with bMF1 via two parts: an N-terminal region with an unstructured loop and a short helix (1–20), and a central long helix (21–60)  and IF1(Δ1–22) maintained inhibitory potency to halt the catalysis and rotation of bMF1, although these mutants were remarkably less effective than IF1(WT) or other mutants. IF1(Δ1–22) was truncated before F22, losing all residues that were in contact or close proximity to the γ subunit. Thus, our study suggests that the interaction with the γ subunit is dispensable, supporting the latter model. Our result was also supported by the research using F1 and IF1 from yeast mitochondria: deletion of all the residues preceding F17 in yeast IF1, corresponding to F22 in bovine IF1, still maintained the inhibitory capacity67. This result implies a universal IF1 inhibition mechanism beyond the species level.Structural analyses of the 60) Fig. a, b. ThebMF1-IF1 and bMF1-(IF1)3, hydrophobic residues in the central long helix, including Y33, form strong interactions with the C-terminal domain of the β subunit, providing most of the binding energy. The binding of IF1 to F1 is further augmented by salt-bridge formation between E30 in IF1 and R408 in the βDP subunit. The contribution of these residues was experimentally confirmed in mutagenetic approaches48. A possible molecular mechanism for IF1 inhibition is that the tight binding of IF1 with the β subunit prevents conformational transition required for rotary catalysis. There are several highly conserved charged residues in the C-terminus of IF1, and the roles of these residues have not been clarified. As a preliminary trial, we conducted molecular dynamics simulation68 where the γ subunit is forcibly rotated in synthesis direction. The interaction between the charged residues of the γ subunit and negative charge of IF1 was suggested, which is reminiscent of the ionic track69. Based on this observation, we tested the role of these conserved charged residues by selectively substituting five residues with alanine  and the alanine-substitution mutant in the IF1 inhibition assay as well as in manipulation assay. Our experiments suggest that C-terminal charged residues have little impact on the inhibitory mechanism and the rotation-direction-dependent dissociation.In the atomic structures of 1 from F1 , which loses residues in contact with the γ subunit, showed a significantly high activation probability after the forcible rotation of 360°. The important finding in this mutant is that both of the clockwise and counterclockwise rotation resulted in the efficient activation from IF1 inhibition with almost equal probability , IF1(Δ1–7), and IF1(Δ1–12), which showed activation only via clockwise rotation. In the case of IF1(Δ1–19), the asymmetric effect of forcible rotation was retained. However, a higher activation probability is observed. These observations indicate that the N-terminal region of the central long helix plays a crucial role in rotary-direction-dependent activation, and the N-terminal short helix also contributes to this result. The truncated residues in IF1(Δ1–22), in addition to the 1–19 truncation, are G20, A21, and F22. Among them, F22 is bulky compared with the other residues. Further, in the crystal structures of the bMF1-IF1 complex, F22 is in direct contact with I16 of the γ subunit. Considering these points, F22 is likely one of the most critical residues for rotary-direction-dependent activation. Molecular dynamics simulations of the bMF1-IF1 complex would provide more detailed information on the molecular mechanism of rotation-direction-dependent activation.Analyses of N-terminal truncation mutants revealed the molecular mechanism of rotary-direction-dependent dissociation of IF F1 Fig. . IF1 at the N-terminus of the β subunit (hereafter referred to as bMF1) was purified as described previously20. IF11–60 with the linker and mScarlet fused to the C-terminus (referred to as IF1(WT)) was purified as described previously48. The N-terminal truncation mutants and the C-terminal alanine substitution mutant of IF1 were prepared as follows. Fragments of the N-terminal truncated mutants, deleted in the region encoding the corresponding N-terminal region, were generated by PCR using sets of mutation primers. The sequence encoding the C-terminal alanine mutant was amplified by PCR using primers containing mutations. The sequences of the PCR primers are provided in the Source Data file. The resulting PCR products were subjected to gel electrophoresis and purification. The insert plasmid and the vector IF1(WT) plasmid were digested with the same restriction enzymes. The products were ligated and introduced into Escherichia coli JM109. The sequence of the recombinant IF1 plasmid was confirmed using the Fasmac sequencing service . The purified mutant proteins were confirmed by SDS-PAGE and MALDI-TOF/TOF mass spectrometry  , 50 mM KCl, 2 mM MgCl2, and an ATP-regenerating system (0.2 mg/mL pyruvate kinase and 2.5 mM phosphoenolpyruvate) supplemented with 0.2 mM NADH and 50 μg/mL lactate dehydrogenase, as described previously48. Experiments were performed at 25°C, unless otherwise indicated, using a V-660  UV/VIS spectrophotometer equipped with a peltier-type temperature controller (ETCS-761). ATP hydrolysis by bMF1 was initiated by adding purified bMF1 to the basal buffer containing 1 mM ATP. For the reaction to reach a steady state, we waited for 180 s before injecting IF1 into the reaction mixture. After the IF1 injection, the rate of ATPase activity changed. Inhibition by IF1 was quantified by estimating the apparent rate constants for IF1  and IF1(Δ1–22) inhibition  through a biotin-avidin interaction. The protocol was as follows20. A flow chamber (~5 μL) was prepared with two coverslips  with double-sided tape  as a spacer. Purified bMF1 (~500 pM) in observation buffer (50 mM HEPES-KOH (pH 7.5), 50 mM KCl, and 2 mM MgCl2) was gently introduced into the flow chamber and incubated for 5–10 min. After washing unbound bMF1 with more than 50 μL of observation buffer containing ~10 mg/mL BSA, streptavidin-coated magnetic beads , which were pre-centrifuged to allow the use of relatively small beads (~300 nm), were introduced into the flow chamber. Unbound beads were washed with more than 50 μL of observation buffer containing the prescribed concentrations of ATP and/or ADP/Pi. Rotations of the magnetic beads were observed using a phase-contrast microscope  with a 100× objective lens at a recording rate of 30 fps. The rotation assay was performed at 23 ± 2 °C. The ATP-regenerating system (0.2 mg/mL pyruvate kinase and 2.5 mM phosphoenolpyruvate) was added to the solution mixture, except when ADP was used.For the rotation assay, 1 inhibitory state, we identified rotating molecules among the particles attached to the coverslip in the IF1-free solution. Next, a solution containing IF1 in ATP solution or ATP synthesis buffer  was infused into the flow chamber. After rotations for several tens of seconds, all bMF1 molecules stopped rotation , which allowed real-time manipulation with magnetic tweezers. Movies were stored on a computer as AVI files and analyzed using custom-made software.Magnetic tweezers, consisting of two sets of electromagnets, were equipped onto the microscope stage and controlled by custom-made software1-free solution were recorded for more than 10 min per molecule  corresponds to the ADP-inhibited form. These results showed that the time scale for ADP inhibition was 10-30 s under the various conditions tested. Distributions of rotating times before lapsing into the pauses were fitted by a single exponential function, Rotations in the IF1 inhibition. Probability plots versus rotating time were fitted with a single-exponential decay function to estimate the mean rotation time, pause was defined as a pause state for more than 1 s. Except for IF1(Δ1–19) and IF1(Δ1–22), most of the pauses showed extremely long pausing states of more than 480 s, corresponding to IF1 inhibition. A few traces showed relatively short pausing states of ~20 s. They were attributable to ADP inhibition because the timescales of these pauses were mostly identical to the values estimated from the IF1-free solution experiment  and IF1(Δ1–22), rotations were frequently recovered without manipulation, suggesting a reversible inhibition/dissociation mechanism. The histogram of pausing time was fitted by single-exponential decay function for IF1(Δ1–19) to estimate the mean duration time, 1(Δ1–22) analysis, the double-exponential decay function was applied to fit the histogram, estimating 1(Δ1–22) inhibitory states.The experimental procedure was described in the main text (see also Fig. Further information on research design is available in the Supplemental informationPeer review fileReporting Summary


The timing of the first appearance of animals is of crucial importance for understanding the evolution of life on Earth. Although the fossil record places the earliest metazoans at 572–602 Ma, molecular clock studies suggest a far earlier origination, as far back as ~850 Ma. The difference in these dates would place the rise of animal life into a time period punctuated by multiple colossal, potentially global, glacial events. Although the two schools of thought debate the limitations of each other's methods, little time has been dedicated to how animal life might have survived if it did arise before or during these global glacial periods. The history of recent polar biota shows that organisms have found ways of persisting on and around the ice of the Antarctic continent throughout the Last Glacial Maximum (33–14 Ka), with some endemic species present before the breakup of Gondwana (180–23 Ma). Here we discuss the survival strategies and habitats of modern polar marine organisms in environments analogous to those that could have existed during Neoproterozoic glaciations. We discuss how, despite the apparent harshness of many ice covered, sub‐zero, Antarctic marine habitats, animal life thrives on, in and under the ice. Ice dominated systems and processes make some local environments more habitable through water circulation, oxygenation, terrigenous nutrient input and novel habitats. We consider how the physical conditions of Neoproterozoic glaciations would likely have dramatically impacted conditions for potential life in the shallows and erased any possible fossil evidence from the continental shelves. The recent glacial cycle has driven the evolution of Antarctica's unique fauna by acting as a “diversity pump,” and the same could be true for the late Proterozoic and the evolution of animal life on Earth, and the existence of life elsewhere in the universe on icy worlds or moons. The fossil record places the earliest metazoans at 572–602 Ma, whilst molecular clock studies suggest an earlier origination, up to ~850 Ma, during a time punctuated by multiple global glacial events . If animal life did arise before or during these glacial periods it would have faced conditions analogous with modern polar marine habitats, requiring similar survival strategies. Ice dominated systems make some environments more habitable and recent glacial cycles have driven the evolution of Antarctica’s unique fauna, the same could be true for the late Proterozoic and the evolution of animal life on Earth. Today, the melting of sea ice in spring and early summer seeds the water with microalgae and microbes  and the ice or crystal krill  feeding on bacteria, diatoms, detritus, and other microorganisms, including the algae that form on the underside of sea ice  exceeding 700 individuals m−2, stalked barnacles, limpets, gastropods, anemones, and a predatory sea star , most of the Antarctic continental shelf is believed to have been covered in thick ice sheets extending almost out to the shelf break  would have likely retreated to the deep during a snowball or partial‐snowball Earth, potentially colonising the underside of the ice or deep water refugia, oxygenated via the sinking of cold surface waters formed during sea ice formation or subglacial melting. Furthermore, ice‐induced disturbances may have driven speciation through fragmentation and subsequent recolonization. We conclude that to elucidate the origins of animals we need to understand recent polar biology and apply this knowledge back to the Neoproterozoic.


The primary outcome was major adverse cardiovascular events . The median follow-up time was 5.24 years. The incidence of major adverse cardiovascular events was 43.2%; patients with dilated LVNC had a higher risk  than those with isolated LVNC. None of the isolated LVNC patients had cardiovascular deaths or severe ventricular arrhythmias. The risk of systemic embolism was similar between patients with dilated and isolated LVNC. Our findings indicate that transthoracic echocardiography is a useful tool for classifying LVNC into subtypes with distinct clinical outcomes. Dilated LVNC is associated with a poor prognosis, while the isolated subtype is probably a physiological condition.Left ventricular noncompaction (LVNC) is a heterogeneous cardiomyopathy that can be classified into different subtypes based on morphologic and functional features. However, the prognosis of the dilated and isolated subtypes of non-pediatric LVNC remains unknown. We retrospectively studied 101 patients with LVNC diagnosed at Peking Union Medical College Hospital from 2006 to 2022 using the Jenni criteria of transthoracic echocardiography. The patients were grouped into those with dilated LVNC ( Left ventricular noncompaction (LVNC) is a rare and unclassified cardiomyopathy characterized by two distinct layers of myocardium with compacted and noncompacted regions, prominent trabeculae formation, and deep intertrabecular recesses ,2,3. EchMYH7, TTN, and MYBPC3, are the most common abnormalities observed, and these mutations frequently overlap with those seen in dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy  and lower LVEF  according to the stratification criteria, but they also presented with more cases of LA enlargement  and more severe mitral regurgitation (moderate to severe) (42.2% vs. 2.7%); these could be explained as the consequences of LV dilation. A total of 93.8% of the patients with dilated LVNC, but only 37.8% of those with isolated LVNC, had BNP/NT-proBNP elevation (p < 0.001). The pre-existing HF seemed not to be enough to explain the difference in the proportions of BNP/NT-proBNP elevation and NYHA III/IV. And we proposed additional explanations that in patients with dilated LVNC, the increased ventricular end-diastolic pressure and wall tension due to cardiac dilation could stimulate natriuretic peptide secretion, resulting in generally elevated BNP/NT-proBNP levels, and that when hospitalized for HF, the symptoms of dilated LVNC patients (more often NYHA III/IV) were usually more severe than isolated LVNC patients. No difference was observed in the lipid profiles between the two groups of patients. A comparison of the baseline characteristics between the patients who were followed up and those who dropped out revealed no significant differences (p = 0.02) was the only definite risk factor for dilated LVNC among the demographic and clinical characteristics included in our study . The results of the univariate and multivariate Cox analyses of variables associated with MACE are presented in p < 0.01). Model II, which was further adjusted by common clinical characteristics associated with cardiovascular events , showed an HR of 6.93 . In the final Model III, which included the results of other examinations , dilated LVNC showed a significantly increased risk of MACE compared with the isolated subtype, with an HR of 4.43 . No other TTE parameters were included in the multivariate regression model; the definitions of dilated and isolated LVNC were already based on a composite pattern of the LVEDD and LVEF, which had strong correlations with other TTE parameters included in our study.Kaplan–Meier curves for MACE and HF stratified by dilated LVNC and isolated LVNC are plotted in p = 0.02). Although a multivariate subgroup analysis of cardiovascular mortality could not be performed because of the limited number of events, an increased risk of cardiovascular mortality in patients with dilated LVNC and a relatively favorable long-term prognosis for isolated LVNC could be predicted. Two non-cardiovascular deaths occurred: one patient with dilated LVNC died of pancreatic cancer, and the other with isolated LVNC died of hepatic cancer. After taking these two cases into account, the incidence of overall mortality was also significantly higher in dilated LVNC (p = 0.04).Kaplan–Meier curves for cardiovascular mortality, systemic embolism, all-cause mortality, and new-onset or worsened arrhythmia between the two LVNC subtypes are plotted in p < 0.01). In the multivariate analyses, after stepwise adjustment by age and sex in Model I , further adjustment by smoking and NYHA functional class in Model II , and final adjustment by ECG findings and BNP/NT-proBNP concentrations in Model III , the association of the dilated subtype with increased HF outcomes was still significant, while none of the other variables included in the regression model could partially explain the distinction of HF occurrence between the two groups.Univariate and multivariate analyses of factors associated with the risk of HF showed results consistent with those of MACE . The Kapp = 0.26), probably because a great portion of arrhythmia events had already occurred before the diagnosis of LVNC and were not reported as clinical outcomes in the follow-up procedure. The number of systemic embolism events was 6 of 54 (11.1%) in the dilated LVNC group and 3 of 34 (8.8%) in the isolated LVNC group. No difference was found in the incidence of systemic embolism using the Kaplan–Meier method (p = 0.53). Because of the limited number of events, multivariate analyses of severe ventricular arrhythmia and systemic embolism could not be performed.Fourteen (15.9%) patients developed new-onset or worsened arrhythmia (incident rate: 3.39 per 100 person-years), including five (5.7%) cases of atrial fibrillation and four (4.5%) cases of implantable cardioverter defibrillator (CRT) defibrillator implantation, which are the most common types of arrhythmia. Both of these severe ventricular arrhythmia events occurred in the dilated group. Among the 14 cases of new-onset or worsened arrhythmia, 10 (18.5%) occurred in patients with dilated LVNC and 4 (11.8%) occurred in those with isolated LVNC. There was no difference between the two groups  isolated LVNC patients with follow-up echocardiography records progressed into the dilated type within 7 years after LVNC diagnosis , while others did not develop cardiac dilation regardless of endpoint events. This suggests that isolated LVNC and dilated LVNC are two distinct subtypes rather than two stages in the natural course of the disease. Therefore, we could conclude that dilated LVNC and isolated LVNC are two clinical entities with different long-term clinical prognoses and that TTE is a simple and effective method to distinguish patients with dilated LVNC who have a high risk of MACE. When a patient’s echocardiography suggests myocardial noncompaction accompanied by LVEF < 45% as well as left ventricular dilation, even though the patient’s current symptoms might not be severe, his future cardiovascular risk is high, and more concerns about drug or implantable device therapy should be addressed by the physician. The results of our study also support the opinion that LVNC is more likely to be a cardiac anatomical variation because a low incidence of cardiovascular events was observed in the isolated LVNC group, and excessive examinations and medications might not be necessary for these patients.The main limitation of this study is that LVNC is a rare cardiomyopathy. This hindered the sample size and limited the number of variables in the regression model, preventing a subgroup survival analysis. Moreover, the retrospective nature of the study had inevitable limitations, such as patient dropout; however, we were able to control missing data in our long-term study to some extent (12.9%), and the demographics of the patients were similar between the two groups. Another limitation is that we only considered the natural progression of the disease. Any interventions through medications such as anti-HF or anticoagulant therapy (which has improved over the last decade) were not considered and could have had an influence on the results. Finally, our research concentrated only on the correlation between phenotype and clinical consequences. More genetic assessments are required to compare genetic variations between different subtypes of LVNC to determine whether the heterogeneity of LVNC has a genetic foundation.Non-pediatric patients with LVNC can be classified into subtypes with different prognoses based on TTE structural and functional parameters. Dilated LVNC is significantly associated with an increased risk of MACE, while isolated LVNC has a favorable prognosis, with no cardiovascular deaths observed in long-term follow-up.


Enhancing the design of bone screw head sockets to prevent stripping and improve the torque required for smooth unscrewing is a significant challenge in orthopedic applications. This research aims to establish a quantitative methodology by integrating mechanical testing with finite element (FE) simulations to determine a safe limitation depth for the screwdriver when engaging with the hexagonal socket, thus avoiding stripped screw heads. A FE model was developed to investigate the biomechanical responses of the screw head design. Five custom-made hexagonal sockets were manufactured, and single load torsional tests were conducted to assess the mechanical performance of the screws and drivers. The results from the mechanical tests were compared with the FE simulations, demonstrating a close agreement and confirming the model’s validity. Furthermore, additional FE models were created to study the impact of manufacturing tolerances on the socket width and screwdriver width. The findings revealed that the maximum torque to failure for the four designs was lower than the margins specified in ISO 6475. Additionally, increasing the depth of the screwdriver led to higher maximum torque values. This research suggests that the technique of screw insertion, specifically the depth of the driver tool within the screw socket, holds greater importance in preventing stripped screw heads than the design and manufacturing width of the bone screw’s hexagonal socket and screwdriver. This confirms the importance of screwdriver engagement inside the bone screw socket to prevent stripped screw heads and sheds light on the added value of maximum torque prediction for future design modifications. Bone screws are the most frequently used implants in orthopedic procedures, whether they are used to properly secure fixation devices to bone or to fix the bone in place and promote its natural healing ,2,3. TheIn current clinical practice, the approximation of the torque in non-locking screws (screws without a locking head system) continues to rely on the user’s intuitive assessment of the required force to attain optimal fixation ,12. ExisDifferent head socket designs are currently employed for both orthopedic and dental screws, including hexagonal, Torx (star shape), and cross sketch configurations . The hexExperimental and clinical studies, using both in vitro and in vivo platforms, can provide relevant information regarding the biomechanical performance of bone screws. However, these studies are generally limited by their inability to estimate the detailed mechanical parameters within the structural components and typically only focus on clinical outcomes. Finite element (FE) modeling offers a potent, cost-effective, and practical approach for conducting non-invasive investigations related to diverse orthopedic implants . By simuTo perform the biomechanical experimental tests, five hexagonal custom-built screw heads and drivers were specially manufactured from a medical grade stainless steel rod (based on ASTM F 138 standard ). The CAThe mechanical performance of the sockets and screwdrivers was evaluated based on torsional tests using a mechanical testing machine  according to ASTM F543 and ASTM F116 ,29 FiguB. A singA nonlinear FE model, developed according to ASTM F543 and ASTM F116 testing techniques, was created to investigate the detailed mechanical responses of the screw head design observed in the experimental test. The implicit model was developed based on the aforementioned manufactured specifications of the socket and screwdriver using ABAQUS  after applying torsional loading according to ASTM F543 and ASTM F116 criteria. The mechanical properties of the model were assumed as nonlinear, isotropic, elastic–plastic and were extracted from the experimental force–displacement data of the medical grade stainless steel rod. A plasticity model, based on the von-Mises yield criterion with hardening rule, was utilized in the simulations, while a large displacement technique with a total Lagrangian algorithm, was used for numerical calculations. The values of the elastic modulus and Poisson’s ratio were 195 GPa and 0.30, respectively. To generate the meshed FE model, 10-node quadratic tetrahedron elements were employed, and comprehensive sensitivity analyses were performed to assess the independence of the results from meshing size and numbers. The final number of elements and nodes used for the socket and screwdriver was 16,079 and 28,797, respectively.The boundary conditions between the socket and screwdriver were determined using the discretization method of surface-to-surface with normal hard contact and tangential contact properties, which allows slippage and separation between the socket and screwdriver during numerical simulations. Coulomb’s tangential contact criterion with a friction coefficient equal to 0.3 was employed ; a torsiUpon FE model validation, different models were developed towards studying the effect of the manufacturing tolerance of the socket width (SW) and screwdriver width (S). Initially, a critical tolerance for the 2.5 mm hexagonal socket and screwdriver was used to generate a model with a maximum socket width tolerance versus a minimum screwdriver width . Next, the critical tolerances were changed while selecting a bigger SW variation as a potential scenario in manufacturing. Considering four different inserted screwdriver depths, 16 different design modes were selected for comparison using the FE analysis as depicted in The 16 FE models with different geometries were then simulated under the torsional loading and boundary conditions previously discussed. The results of maximum torque and stress distribution were analyzed and compared to investigate the influence of screw head design for extracting stripped screw heads. In addition, the slippage-resisting free rotation angles were extracted as an important index to investigate the variations in the ability of resisting cam-out failure for different groups in this study. For this purpose, this geometrical angle was defined as the angle between the lines OA and OB, in which the point O is the center, point A shows the initial contact point, and point B shows the deformed position of point A at the end of simulation .The average maximum torque measured in the experimental tests based on the five samples was 4097.4 (±118.15) N·mm, as compared to the relevant calculated torque of 3919 N·mm obtained from the FE model. Overall, the FE model results were well comparable with the experimental tests , confirmThe simulations for 16 different conditions indicated that the maximum torques to failure for the four designs with the lowest insertion depth  were less than 1000 N·mm, which is less than the margins specified in ISO 6475. By increasing the insertion depth from 0.5 mm to 1 mm, the maximum torque increased on average by 108.52%, and the increase was 353.58% and 442.63% for 1.5 and 1.8 mm insertions, respectively . The resThe von-Mises stress distributions of the maximum torque in the sockets are shown in For different FE simulation results, slippage-resisting free rotation angles were presented in Although the options for operative fracture treatment using metal implants have substantially increased in the last decade, orthopedic surgical implant removal remains challenging and riddled with complications . This isWhile efforts have been made by manufacturers to improve the design of screw heads , sliThis study aims to provide a valid quantitative methodology towards the investigation of bone screw head design for extracting stripped screw heads. Custom-made sockets were designed and manufactured, and single load torsional tests, using a standard mechanical testing machine, were conducted to evaluate the performance of the screws and drivers. The obtained results from the experimental tests were then compared with a nonlinear FE model specifically developed and validated for this purpose. The FE model was created to provide a comprehensive understanding of the intricate biomechanical behavior of the screw head design, based on the standard testing techniques outlined in ASTM F543 and ASTM F116. To ensure the model’s accuracy and reliability, a rigorous validation procedure was employed, comparing the model’s predictions with experimental data from five distinct samples. The validation process yielded promising results, demonstrating an acceptable error rate of less than 15% for all five samples . These oDifferent FE models were consequently developed to investigate the impact of the socket width (SW) and screwdriver width (S) on manufacturing tolerances in relation to screw head design for extracting stripped screw heads. Four different depth insertions  were simulated by way of emulating real-life clinical surgical scenarios. It is noteworthy that during the process of bone healing, it is common for the socket to be filled with ingrown tissue, which can create a challenge where the screwdriver is unable to fully penetrate the socket’s depth. This condition can affect the stability and functionality of the bone screw. On the other hand, the findings of this study reveal that the impact of screwdriver depth on the maximum torque is more significant than variations in the width of the hexagonal socket (SW) and screwdriver (S). This suggests that adjusting the depth of the screwdriver has a greater influence on the maximum torque than modifying the dimensions of the hexagonal socket and screwdriver width. Therefore, it is recommended to ensure an appropriate depth of the screwdriver as a critical factor for achieving optimal torque and implant stability, even in cases where ingrown tissue might impede the complete insertion of the screwdriver into the socket. Highlighting the relative importance of screwdriver depth over SW and S width variations provides valuable insight in terms of considering the depth factor during surgical procedures and implant placement to optimize the biomechanical performance and long-term success of the implant. The effect of socket shape is another important factor  which waIn accordance with ISO 6475, the minimum breaking torque values are established for different types of screws based on the nominal thread diameter . For exaIn socket–screwdriver constructs, interfacial misfit is an inherent consequence of manufacturing tolerances and design parameters ,32. The Certain limitations in the current study should be deliberated here. The first is related to modeling the socket and screwdriver while neglecting the complete bone fixation system, including the bone tissue. This simplification was adopted to focus on the main objective of this study and reduce the computational cost, although future work can benefit from a more inclusive model. The second limitation lies in applying pure torsional torque without consideration of compressive loading during implantation. Although this is a common simplification in related studies in which experimental protocols are designed based on the aforementioned standards, a supplementary study to evaluate the results for combined loading would enhance the findings; it could also be coupled with the current FE methodology for future work. Furthermore, the ultimate failure torque may be slightly higher than the highest achievable torque during manual use for ordinary screwdrivers in surgery. However, the results achieved in this investigation can be regarded as a valuable guideline for approximating the maximum torque in bone screw applications. The outcomes of this work provide insight into the biomechanical interaction of the socket and screwdriver engagement and may serve as a basis for informing future design modifications aimed at optimizing their performance. By utilizing the results as a reference point, researchers and designers can assess the potential impact of various modifications to bone screw heads on their torque-bearing capacity, including improved screw head designs with enhanced strength and stability.Despite the common use of bone screws and the frequently associated complications in orthopedic implant surgeries, there remains a gap in the literature regarding the influence of manufacturing tolerances for the augmentation of the conventional hexagonal socket design. The main contribution of this work lies in providing a modular FE-based methodology for identifying the safe limitation depth of the screwdriver while engaging with the hexagonal socket to avoid stripped screw heads. This study also emphasizes the relevance of the combination of an adequate depth of the screwdriver with optimal screw head socket and driver designs towards estimating the maximum torque-slippage value and preventing screw slippage. The results confirm that the screwing technique, or depth of the driver tool inside the screw socket, is more important than the design and manufacturing width of the bone screw hexagonal socket and screwdriver. This work also emphasizes the importance of screwdriver engagement inside the bone screw socket, while shedding light on the value of maximum torque prediction for future design modifications. Finally, understanding the relationship between misfit, insertion depth, and slippage-resisting free rotation angles allows researchers and manufacturers alike to refine design configurations, parameters and processes towards enhanced safety, reliability, and effectiveness of orthopedic screws.