In late 2019, a new coronavirus (CoV) caused the outbreak of a deadly respiratory disease, resulting in the COVID-19 pandemic. In view of the ongoing pandemic, there is an immediate need to find drugs to treat patients. SARS-CoV-2 papain-like cysteine protease (PLpro) not only plays an important role in the pathogenesis of the virus but is also a target protein for the development of inhibitor drugs. Therefore, to develop targeted inhibitors, it is necessary to analyse and verify PLpro sites and explore whether there are other cryptic binding pockets with better activity. In this study, first, we detected the site of the whole PLpro protein by sitemap of Schrödinger (version 2018), the cavity of LigBuilder V3, and DeepSite, and roughly judged the possible activated binding site area. Then, we used the mixed solvent dynamics simulation (MixMD) of probe molecules to induce conformational changes in the protein to find the possible cryptic active sites. Finally, the TRAPP method was used to predict the druggability of cryptic pockets and analyse the changes in the physicochemical properties of residues around these sites. This work will help promote the research of SARS-CoV-2 PLpro inhibitors. In late 2019, a new coronavirus (CoV) caused the outbreak of deadly respiratory disease, and the COVID-19 pandemic brought major harm and challenges to more than 200 countries and regions around the world . Due to Inhibitors targeting SARS-CoV-2 PLpro are excellent candidates for antiviral drug development, as they not only block virus replication but also inhibit the dysregulation of signaling cascades in infected cells . UnderstAccording to previous literature reports and information from the Protein Databank (PDB), there are several known SARS-CoV-2 PLpro targets, a canonical cysteine protease catalytic triad site  ,20,21, aThe set of amino acid residues around a binding pocket determines its physicochemical characteristics and, together with its shape and location in a protein, defines its functionality. The mobility of proteins allows the opening, closing, and adaptation of binding pockets to regulate binding processes and specific protein functionalities . Many prThe research workflow is shown in Through the three static site detection methods, namely, Schrödinger, LigBuilderV3, and DeepSite, we obtained the following results. The site detection and scoring of different conformations by the three methods are shown in We used MixMD Probeview to analyse the occupancy grids and obtained the hotspot ranking . The firIn the NMA MixMD system, NMA probe molecules have the smallest peptide bond and can simulate the role of peptide substrates in cryptic pockets. In the probe distribution density ranking a, the fiIn the IPA MixMD system, IPA probe molecules can easily capture small hydrophobic interactions and provide hydrogen bond receptors and donors. In the molecular density ranking of the probe a, the fiFor the PHN MixMD system a, PHN prFor the PYR MixMD system a, PYR prWe found that there were many overlapping points in the simulated trajectories of different mixed probe molecules. We counted and ranked these highly overlapping points , includeWe simulated the mixed solvent molecular dynamics, established the solvent system of five mixed probe molecules, attempted to use five probe molecules to induce the conformational change of PLpro, and then attempted to locate the cryptic active pocket. In this study, the stability of six model systems was verified according to the molecular dynamics trajectory of the simulated system. The root-mean-square fluctuation (RMSF) of each amino acid residue and the root-mean-square deviation (RMSD) between the amino acid heavy atom near the protein main chain and the initial conformation were calculated mainly based on the analysis program of the GROMACS 2018.7 software . We analThe RMSF results  showed tBy using the TRAPP method, we dynamically analysed these main sites: catalytic triad site, thumb site 1, thumb site 2, finger site, and back site 1. First, we used the TRAPP-LR/CNN score curve to evaluate the druggability of each site. Second, we analysed the obvious properties changes of each site. Third, we analysed the associated residues at each site. Finally, we analysed the conformation after clustering and compared it to explore the main factors affecting the druggability of this site.For the catalytic triad site, according to the molecular density ranking of the probe, we selected the NMA mixed solvent system trajectory for comparison with the pure water system trajectory. It can be seen from For thumb site 1, we selected the IPA mixed solvent system trajectory to compare with the pure water system trajectory. In In addition to the above two known sites, we also analysed the other three possible cryptic sites. For thumb site 2, we selected the ACN mixed solvent system trajectory for comparison with the pure water system trajectory. In both the CNN and LR model curves , the druFor the finger site, we selected the PHN mixed solvent system trajectory to compare with the pure water system trajectory. Through observing the score results curve , in the For back site 1, because the ranking of NMA, ACN, and PYR is the highest in the probe distribution, we selected these three groups of mixed solvent system trajectories to compare with the pure water system trajectory. According to the scoring of the four systems , from thUsing the TRAPP method, we analysed the change in the physicochemical properties of the residues around each active binding site in different systems. From the overall druggability scoring curves of the TRAPP-LR/CNN models , the resIn summary, for the catalytic triad site, we found a pocket volume that restricts its pocket druggability, which was not improved by the insertion of NMA probe molecules. For thumb site 1, we know that the insertion of IPA probe molecules has a positive impact on the druggability and related physical and chemical properties, and, among the five sites analysed, the result is the best from the druggability scoring curve of the overall trajectory. For the two known binding sites, through the above results, we know that the physical and chemical properties of thumb site 1 are more likely to be changed after being induced by the probe molecules to increase the druggability. However, at present, most studies mainly focus on the catalytic triad site. In the future, it may be necessary to strengthen the research on thumb site 1, hoping to find better small molecule inhibitor drugs for SARS-CoV-2 PLpro. Thumb site 2 may be the next SARS-CoV-2 PLpro potential cryptic binding site. At the same time, we also found that the insertion of the ACN probe molecules has a positive inducing effect on the improvement of the druggability of thumb site 2.The initial X-ray crystal structures of SARS-CoV-2 PLpro were downloaded from the Protein Databank (PDB), PDB ID : 6WUU 6, 7D47 . First, the downloaded protein file (6WUU.pdb) was prepared by Schrödinger’s protein preparation wizard (version 2018), which removed excess water molecules and heteroatoms from the protein, built the missing side chain atoms, corrected the structure of the protein, and added hydrogen atoms. Then, we run the MD for the six systems, single water of 6WUU, and mixed solvent  of 6WUU with GROMACS (version 2018.7) [We download the initial X-ray crystal structures of SARS-CoV-2 PLpro from the Protein Databank (PDB), PDB ID: 6WUU. The probe molecules of IPA, ACN, PYR, PHN, and PYR were obtained from Ligand Expo web ( 2018.7) . The sysFollowing simulation, 1 µs of each MixMD trajectory was aligned and generated in a 0.5 Å × 0.5 Å × 0.5 Å grid using the CPPTRAJ module in AmberTools21. Load files to be analysed into PyMOL-MixMD Probeview. MixMD Probeview was used for the analysis of the occupancy grids and obtained the hotspot ranking and to compare the position detection results obtained in the previous target analysis and delete the pockets with low druggability.In the six systems, single water, and five mixed solvents, a 100 ns trajectory was taken. For different binding sites, according to the density ranking of probe molecules, we selected the probe mixed solvent system with the highest ranking at the corresponding points to compare with the pure water solvent system to observe the effect of the probe molecules on sites. According to the pocket position, the residues around the binding site are selected. We selected the residues of the catalytic triad site, thumb site 2, and finger site and back site 1 within 10 Å and the residues of thumb site 1 within 8 Å as the comparison objects . This prThe grid contains N grid points in one channel. A grid point is denoted as Currently, the COVID-19 pandemic has become a major global public health crisis. In view of this situation, there is an urgent need to find drugs to treat patients. The papain-like protease domain of SARS-CoV-2 nsp3 has emerged as a viable drug target for the development of anti-SARS therapeutics. Primarily, through three protein site detection methods, sitemap of Schrödinger, cavity module of LigBuilderV3, and DeepSite, we verified the known SARS-CoV-2 PLpro targets, namely, catalytic triad site, thumb site 1, and palm site, and tested the reliability of these methods and found several regions that may have cryptic sites. Subsequently, through MixMD and Probeview procedures, we obtained the molecular density ranking of the probe, made site statistics for each mixed system, found some highly overlapping sites, compared the previous site analysis of the original structure, and then identified the three most likely cryptic sites: finger site, thumb site 2, and back site 1. Next, we used the TRAPP method to analyse the known sites and possible cryptic binding sites, as well as the various druggability related attributes of the residues around the site. Eventually, through a series of analyses and comparisons, we found that thumb site 2 has a certain development potential, and the in-depth study of this site will promote the development of inhibitor drugs against SARS-CoV-2 PLpro. In summary, we hope that this paper will promote drug design and discovery against COVID-19.


In recent years, the development of wireless technologies has led to fast growth in mobile networks, especially with the rise of 5G New Radio (5G NR). A huge number of base stations (BSs) are mandatory to serve the growth of mobile services, which has led to concerns about the increase in electromagnetic field (EMF) radiation exposure levels. To control the overall power emitted by EMF transmitters, international bodies have set maximum exposure limits. This paper investigates the compliance distances (CDs) of shared sites by a group of Mobile Network Operators (MNO) as multi-operators operating with multi-technology and sharing the same tower. The study investigated the CDs of the most two commonly used types of sharing sites, macro and indoor-Based solution sites (IBS). In addition, the study analyzed the power densities and total exposure ratios for the general public and occupational workers in each sharing scenario. The results showed that, compared with a single MNO, the CD increased by 41% in the case of two MNOs, 73% for three MNOs, and 100% for four MNOs. The EMF site sharing scale-up formula was used to estimate the increase in CDs for N number of MNOs assuming that all MNOs use the same site configuration. In addition, the results showed that 5G has the highest contribution to the total exposure ratio (TER) at the CD in the main direction of the antennae. Mobile networks are the fastest-growing wireless communication networks due to the massive increase in data service consumption, especially with the wide spread of smartphones and tablets. This leads to the generation of large data traffic over the mobile network that will reach very high figures by 2030 compared with 2020 ,2. ExpanIt is predicted that 5G technology will be the system for general purposes  because It is less expensive to share a site than to build a new one. According to , infrastThe densification of the existing sites increases the difficulty of acquiring more physical sites within the required nominal locations and leads to fewer options.Site sharing enables the rationalization of the legacy 2G and 3G networks, taking into account the declining revenues from 2G and 3G networks and the higher spectral efficiency of the next generation of 4G and 5G technologies.Site sharing enables diverting the investment to other important innovations, such as the deployment of more 5G sites.Site sharing has some social benefits because it lowers network costs, which lowers the customer’s service fee.Tower sharing benefits the environment by reducing the number of sites with better looks and views.In many countries, the MNOs have started to share mobile sites to expand their network services in terms of coverage footprint and capacity resources . Site shNetwork sharing can proceed in many ways, starting from a small part of the infrastructure up to full sharing, including core parts . SharingThe second group is active sharing, which consists primarily of sharing the Radio Access Network (RAN) component with a dedicated frequency spectrum for each operator as the Multi-Operator RAN (MORAN) and a shared frequency spectrum as the Multi-Operator Core Network (MOCN), as shown in Passive site sharing raises concerns about the total EMF exposure radiated from the antennae because each operator has separate radio equipment and all of them transmit from the same location. In this study, we determined the total accumulated exposure by calculating and analyzing the CDs for the two types of site sharing at the macro site and the indoor IBS site.The merit of this analysis is in the calculations that consider the real operating setup  and involve the actual realistic transmitted power. Additionally, the goal was not to compare the levels of exposure to different technologies. Instead, it was to find out what the CDs are when different technologies and operators all transmit in the same place.This manuscript is structurally organized into three main sections: the exposure standard limits, CD calculations, and the results and discussion for site sharing at macro and indoor sites.The guidelines of the ICNIRP are the most widely adopted and used for controlling non-ionizing radiation exposure in many countries ,20,21. Tif is the frequency range, The ICNIRP also specified the total reference limits from instantaneous concurrent exposures from multiple sources, which are aggregated using Equation (1) shown below :(1)∑i=10CD) to refer to the distance from the antenna at which TER = 1, which means that the accumulated exposure reaches the maximum limit. The TER was calculated on the basis of the power density Sinc; therefore, Equation (2) was modified to Equation (3), as shown below [In , the Intwn below .(2)TER=inc is the power density, which can be expressed as:At distance R (m) from the antennae, the SEquation (3) can be substituted in the nominator of Equation (2), so the TER would be:CD, the TER = 1, then:At compliance distance RWith the introduction of 5G NR, which uses a high grade of massive Multi-Input Multi-Output (mMIMO), some recent studies have investigated EMF exposure by considering more factors ,30,31,32A modern mobile wireless network is comprised of a combination of different site types and different access technologies that work together as a heterogeneous network. The RF planning teams in the MNOs select the appropriate site type according to many inputs, such as the target coverage range, number of users, forecasted traffic growth, total cost, and nature of the area . This study looked into multi-operator, multi-technology CDs for two types of sharing sites, macro and indoor IBS sites, which are currently the most commonly used.The macro site is a general solution that is used by all operators to provide coverage in populated areas . The macTypical configurations of macro sites equipped with multi-technology solutions were used as input data for the calculations. The site was operated with six technologies: 2G GSM at 900 MHz (G900), 3G UMTS at 900 MHz (U900), 4G LTE at 800 MHz (L800), 4G LTE at 1800 MHz (L1800), 4G LTE at 2100 MHz (L2100), and 5G NR at 3500 MHz (N3500). The input data for each technology, including Tx/Rx, antenna gain, frequency bandwidth, system load, and transmitting power, are listed in CD (RDC-H and RDC-V), as displayed in DC-H distances were longer than the vertical RDC-V distances, and the ICNIRP has a little more restricted RCD compared with the FCC. In the main antenna horizontal direction, the general public ICNIRP RCD-H was 14.81 m when there was a single MNO, while it increased to 29.6 m when 4 MNOs shared the macro site. The general public FCC RCD-H was 13.8 m when there was a single MNO, and it increased to 27.7 m for 4 MNOs. CD in both the vertical and horizontal directions for the GP.The site configuration data were utilized in Equation (6) to compute the vertical and horizontal R2) calculated at distance (m) from the antennae in the presence of four MNOs, and the analysis showed that 5G N3500 had the highest PD among the technologies, and all technologies reached high levels at distances very close to the macro site’s antenna (less than 3 m).Further processing was carried out to analyze the total emitted power density and related contribution to the total exposure ratio. With reference to the ICNIP for the GP, CD with 34%, followed by 4G L800 with 24%, 4G L1800 and L2100 with 9–10% each, and 2G G900 and 3G U900 with 11% each. Similarly, at the RCD based on FCC limits, the highest contributor was N3500 (39%), followed by L800 (21%), L1800 and L2100 (11% each), and G900 and U900 (9% each). CD, and these were in line with the results found in [The results showed that 5G N3500 had the highest contribution at the Rfound in ,37 for tCD increased with the increase in the number of MNOs sharing the same location; Equation (6) gives shorter compliance distances compared with the calculations performed using Equation (4) because it is based on realistic rated power for the mMIMO rather than the conservative power levels used in Equation (4). The RThe results in For N number of MNOs, Equation (7) becomes Equation (8):Equation (9) represents a simplified formula that gives the compliance distance based on the EMF Site-Sharing Scale (EMF-SSS formula). In reality, it is very hard to find a group of MNO operators that use the same configuration for multi-technology, including the power rating and system load. However, Equation (9) is useful for predicting the CD and indicating the expected increase in the CD when accommodating more than one MNO at the same site, and Most of the traffic in mobile networks is generated from indoor houses and buildings , and in The IBS-DAS consists of many connected antennae that are shared and energized from the same radio transmitters in the splitting system. In this study, we looked at radiation from a single antenna (one zone) using high-density configurations of a single IBS site outfitted with multi-technology. The IBS was operated with six technologies: G900, U900, L800, L1800, L2100, and N3500. The input data for each technology are listed in DC-H distances were longer than the vertical RDC-V distances, and the ICNIRP had a slightly more restricted RCD compared with the FCC. The general public ICNIRP RCD-H was 0.89 m when there was a single MNO, but it increased to 1.74 m for four MNOs. Additionally, for the general public, RCD-H was 0.4 m when there was a single MNO, while it increased to 0.79 m for four MNOs. CD for the general public in both the vertical and the horizontal direction.Similar to the macro results, the IBS results expressed that all R2) calculated at a distance (m) from the antennae, and the analysis showed that 5G N3500 had the highest PD compared with the other technologies; it reached high levels at distances very close to the DAS antenna.In addition, further processing was carried out to analyze the power density and its related contribution to the total exposure ratio. With reference to the ICNIP for the GP, CD, and these were the expected results because the IBS site does not use a high grade of mMIMO.The results showed that 5G N3500 MHz had the highest contribution to the TER at the compliance distances for the ICNIRP with a contribution of 26%, followed by 4G L800 with 20%, 2G G900 and 3G U900 with 18% each, 4G L1800 with 9%, and 4G L2100 with 8%. Similarly, the N3500 had the highest contribution (30%), followed by 4G L800 (18%), 2G G900 and 3G U900 (16% each), and 4G L1800 and 4G L2100 (both with 10%). CD of the IBS increased with the same percentage for all references. Similar to that of the shared macro site, with an increase in the number of MNOs, the RCD with the increased N of MNOs sharing the IBS, as shown in Additionally, in practice, it is difficult to find groups of MNO operators that share IBS sites with the same configurations. However, using CD distances were 14.8 m and 13.8 m according to the ICNIRP and FCC, respectively. Furthermore, for a macro site shared by four MNOs, the RCD distances were 29.61 m and 27.69 m according to the ICNIRP and FCC, respectively. In addition, for all shared macro site scenarios, the 5G N3500 MHz had the highest contributions to the TER of 34% and 39% at the horizontal RCD according to the ICNIRP and the FCC, respectively. For the indoor IBS site with a single MNO, the general public’s horizontal RCD distances were 0.87 m and 0.8 m according to the ICNIRP and FCC, respectively. In addition, for an IBS shared by four MNOs, the RCD distances were 1.74 m and 1.6 m according to the ICNIRP and FCC, respectively. Moreover, for all shared IBS site scenarios, the 5G N3600 MHz had the highest contributions to the TER of 26% and 30% at the horizontal compliance distances according to the ICNIRP and FCC, respectively. In the future, the research team will investigate the RCD for the accumulated radiation considering the neighboring sites. The EMF exposure compliance boundary for multi-technology, multi-operator site sharing was discussed and analyzed using calculations that considered realistic configurations, and the study covered two types of site sharing: macro and indoor IBS sites. For a typical site operating with 2G/3G/4G/5G, the compliance distance results for the general public zone were less than those for the occupational zone. The ICNIRP limits were slightly more restricted than the FCC limits. For a macro site with a single MNO, the general public’s horizontal R


In 48 h, the induced serine/threonine-protein kinase and CDK12 in worker embryos signify their roles in the construction of embryonic tissues and organs; however, the highly activated kinases CDK1, raf homolog serine/threonine-protein kinase, and MAST2 in drone embryos may drive the large-scale establishment of tissues and organs. In 72 h, the activated pathways and kinases associated with cell growth and tissue differentiation in worker embryos may promote the configuration of rudimentary organs. However, kinases implicated in cytoskeleton organization in drone embryos may drive the blastokinesis and dorsal closure. Our hitherto most comprehensive phosphoproteome offers a valuable resource for signaling research on phosphorylation dynamics in honey bee embryos.Protein phosphorylation is known to regulate a comprehensive scenario of critical cellular processes. However, phosphorylation-mediated regulatory networks in honey bee embryogenesis are mainly unknown. We identified 6342 phosphosites from 2438 phosphoproteins and predicted 168 kinases in the honey bee embryo. Generally, the worker and drone develop similar phosphoproteome architectures and major phosphorylation events during embryogenesis. In 24 h embryos, protein kinases A play vital roles in regulating cell proliferation and blastoderm formation. At 48–72 h, kinase subfamily dual-specificity tyrosine-regulated kinase, cyclin-dependent kinase (CDK), and induced pathways related to protein synthesis and morphogenesis suggest the centrality to enhance the germ layer development, organogenesis, and dorsal closure. Notably, workers and drones formulated distinct phosphoproteome signatures. For 24 h embryos, the highly phosphorylated serine/threonine-protein kinase minibrain, microtubule-associated serine/threonine-protein kinase 2 (MAST2), and phosphorylation of mitogen-activated protein kinase 3 (MAPK3) at Thr Worker and drone bees are distinguishing castes in the honey bee colony. They are derived from fertilized (diploid) and unfertilized (haploid) eggs laid by queen bees . ThroughEmbryogenesis is a complex rudimentary organ-generating process involving nuclear cleavage (<10 h), blastoderm formation (∼20 h), germ band differentiation (∼48 h), and dorsal closure (∼60 h), which occurs for about 72 h in both worker and drone bees . Due to The reversible phosphorylation of serine, threonine, and tyrosine residues is a critical regulatory mechanism that allows spatial and temporal regulation of the activation states and conformations of proteins, thereby regulating diverse downstream effects . High-reDrosophila. For example, the phosphorylated ubiquitin carboxyl-terminal hydrolase is associated with compound eye formation and oocyte development (2), can yield significant expansion of phosphoproteome coverage. This is of paramount importance for phosphoproteome studies.Phosphorylation contributes to the regulation of tissue configuration during the process of morphogenesis, during which the rudimentary organs are formed. Here, many phosphoproteins in the honey bee embryo may function as in elopment , and theelopment . Serine/elopment . Broad-celopment . PhosphoAlthough proteome drives the embryogenesis , and the2, Titansphere, 5 μm particles) was bought from GL Science . Ti4+-IMAC material was obtained from Dalian Institute of Chemical Physics, Chinese Academy of Sciences.Unless the source is otherwise specified, all chemicals were purchased from Sigma-Aldrich . Titanium dioxide (TiOApis mellifera) colonies used for sampling were raised at the Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing, China. The worker and drone eggs from five colonies were sampled at 24, 48, and 72 h of age after being laid by the queens in worker or drone combs (for 3 h) based on our previous works  and iodoacetamide . The sample was digested in solution according to the ratio 1:65 (enzyme: protein). After that, the digested samples were divided into two equal parts for phosphopeptide enrichment by TiO2 and Ti4+-IMAC. For phosphopeptide enrichment by TiO2 resin, the digested samples were reconstituted in 500 μL of the binding solution, 6.0% trifluoroacetic acid (TFA)/80% acetonitrile (ACN)/0.2 M dihydroxy-benzoic acid (DHB). A TiO2 slurry was prepared at a concentration of 10 mg/ml in a binding solution. About 50 μl of the prepared TiO2 slurry (10 mg/ml in binding solution) was added to the above mixture and incubated at RT for 60 min under vigorous shaking. Then, the supernatant was discarded, and the resin was washed sequentially with 200 μl of the binding solution, containing 0.5% TFA/50% ACN and 0.1% TFA/30% ACN. The phosphopeptides were eluted from the resin twice with 100 μl of a 0.5 mM K2HPO4. Each procedure of washing, and elution was rotated for 15 min at RT. The elutes were desalted by Zip-Tip C18 columns , vacuum dried, and stored at −80°C for further LC−MS/MS analysis.To attain a deeper phosphoproteome coverage, the phosphopeptides in the worker and drone embryos were enriched by both TiO4+-IMAC, the immobilized Ti4+ polymer beads were prepared by overnight incubation of 10 mg in 100 mM Ti(SO4)2 solution at RT under gentle stirring. The Ti4+-IMAC beads were centrifuged at 20 000×g for 2 min to remove the supernatant, followed by washing with distilled water several times to remove the residual titanium ions. Finally, the obtained Ti4+-IMAC beads were dispersed in 30% ACN containing 0.1% TFA before usage. The phosphopeptide enrichment was carried out as described above in TiO2 enrichment.To enrich phosphopeptides by TiThe enriched phosphopeptides were resuspended in 0.1% FA and analyzed by an Easy-nLC 1000  coupled to a Q-Exactive MS . The peptides were loaded onto a trap column  for 2 min in buffer A (0.1% formic acid) at a flow rate of 5 μL/min. The trap column effluent was transferred to a reversed-phase microcapillary column . Reversed-phase separation of the peptides was done chromatographically with a 120-min gradient from 8% to 30% acetonitrile in 0.1% formic acid with a flow rate of 350 nl/min.https://proteomecentral.proteomexchange.org) via the iProX partner repository . The MS was run in positive ion mode, implementing dynamic exclusion. The MS continuously collected spectra in a data-dependent way over the entire gradient. MS1 precursor scan (m/z 350-1600) acquisition was performed in the Orbitrap using a nominal resolution of 70,000 at m/z 400. The top 20 highest intensity multiply charged precursor ions were fragmented by higher energy collisional dissociation (HCD), with normalized fragmentation energy of 35%. MS2 scans  were acquired in the Orbitrap mass analyzer using a resolution setting of 17,500 at m/z 400. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (pository  with theApis mellifera (downloaded April 2022 from NCBI) and common contaminants, totaling 21,778 entries. The database search parameters were: precursor ion and MS/MS tolerances: 15 ppm and 0.05 Da; the enzyme specificity: trypsin; the maximum missed cleavages: 2; fixed modification: carbamidomethyl ; and variable modification: oxidation , and phosphorylation . The target and decoy sequence fusion strategy was used to control the false discovery rate (FDR) at 1% at the peptide level for protein identification. Scaffold PTM  was used to assess the localization probability of p-sites according to Ascore algorithm  against the protein sequences of lgorithm . All MS/https://gps.biocuckoo.org)  of A. mellifera from the database of STRING (https://www.string-db.org/). Then by BLAST search, the proteins of PPIs were mapped to the benchmark sequences of the proteome in Apis mellifera L. downloaded from the database of Bee Base    was usedsearches , whereasf STRING  v10 (httion 3.2) . Finallyd ssKSRs . The GPSd ssKSRs . Then thd ssKSRs , the nump-value (one-way ANOVA) were made on the sum of the normalized abundances across all runs . One sample was automatically selected as a reference. Based on an expectation-maximization algorithm, feature detection was employed separately on each sample. A high-performance retention time algorithm was used to align the features of the same peptide from different samples . Peptideall runs . Proteinhttp://www.ici.upmc.fr/cluego/)  term was enriched by ClueGO v2.3.3 (cluego/) , a Cytoshttp://kobas.cbi.pku.edu.cn)   was appl.edu.cn) .15 (CDK1 Y15) and anti-Mitogen activated protein kinase 3 Thr564 (MAPK3 T564), at dilutions of 1:4000 and 1:3000, respectively. The secondary antibody  was horseradish peroxidase-conjugated goat anti-rabbit at a dilution of 1:5000. Samples of 10 μg protein were separated by stacking (4%) and separating (12%) SDS-PAGE gels, and each sample was run in triplicates. Protein was transferred to nitrocellulose membranes. The protein bands were visualized by chemiluminescence and quantified by densitometry using a Quantity-one image analysis system . The protein abundance was normalized by GAPDH. The student t-test was used for statistical analysis of protein abundance.To validate two p-sites in different developmental phases of worker and drone embryos, Western Blotting analysis was done as previously described  using thThe activity of the crucial kinases, CDK5, MAPK3, and CDK1, were measured according to the instruction. The kinase assay kits were bought from MyBiosource, Inc. . Briefly, honey bee embryos were homogenized in ice-cold PBS with a glass homogenizer. The resulting suspension was subjected to ultra-sonication to break the cell membranes further. Then the homogenates were centrifuged for 15 min at 1500 × g. Remove the supernatant and assay immediately. The assay samples and standards were incubated with kinase-HRP conjugate in a pre-coated microtiter plate for 1 hour. After the incubation period, the wells were decanted and washed five times. The wells were then incubated with a substrate for HRP enzyme. The product of the enzyme-substrate reaction forms a blue-colored complex. Finally, a solution was added to stop the reaction, turning the solution yellow. The optical density of each plate was measured with Multiskan GO  at a wavelength of 450 nm. The standard curve is used to determine the activities of samples.To validate the role of the crucial kinase in the phosphorated network, the CAMK2 was selected for RNA interference. The siRNA sequences of CAMK2 were designed according to the technical requirements and then synthesized .To investigate the transcription activity of CAMK2 after RNAi, Real-time qPCR was carried on. Total RNA extracted from the embryo or larva samples harvested after RNAi  was used to generate cDNA using Reverse Transcriptase kit reagents  following the manufacturer’s instructions. The primer pairs are designed and synthesized by BGI , and gly2 and Ti4+-IMAC enrichment, showing excellent technical and biological reproducibility.Three-time points representing significant stages of embryonic development were selected: division of the embryo cell and formation of the blastoderm (0–26 h), shaping of the germ band and germ layer (26–46 h), and organogenesis (46–72 h). Pearson correlation coefficients between replicates of the same samples were above 0.91 , regardl2 and Ti4+-IMAC, of which 734 p-sites overlapped , CDK12  and MARK2  was increased during embryogenesis; the number of pT in CDK2 , CDK1 , mig-15  and CDK12  followed the same trend; pY in CDK2  and CDK1  were the significant p-sites in 48 h embryos .By evaluating the phosphoproteome profile change during the development of the worker embryo, we found 195 phosphoproteins differentially regulated in a total of 2145 phosphoproteins . Among t2 and Ti4+-IMAC, respectively , CDK12 , and AbI  was the most mode of p-sites in the 48 h embryos. The number of pT increased along with the embryonic development in kinases STPK  and CDK7  .Amongst 1423 phosphoproteins, 71 were changed in their expression level during the embryogenesis of honey bee drone , of whicAt 24 h of age, 3114 p-sites from 3015 phosphopeptides in 1254 phosphoproteins were identified (2617 p-sites from 2451 phosphopeptides in 1061 phosphoproteins of worker embryos and 1646 p-sites from 1449 phosphopeptides in 708 phosphoproteins of drone embryos). In worker embryos, the proteins were enriched in the functional groups: positive regulation of catalytic activity and microtubule-based process . In dronIn comparing differences in enriched biological pathways between the embryos of worker and drone honey bees, in 24 h embryos, the pathways of wnt signaling and MAPK signaling was commonly enriched in worker and drone. In worker embryos, spliceosome, endocytosis, and starch and sucrose metabolism were uniquely enriched, whereas, in drone embryos, RNA transport pathways were exclusively enriched .Furthermore, the activity of the critical kinases in the worker and drone embryos was compared. At 24 h, the activity of most kinases was higher in worker embryos, whereas only kinase PKG was high in drone embryos .In comparing site occupancy between worker and drone embryos, we focused on 12 high-activity kinases . NotablyAmong the 1254 phosphoproteins, 84 were differentially regulated, of which 15 and 69 were up-regulated in worker and drone, respectively. The up-regulated phosphoproteins in the drone were enriched in the functional categories: cytoskeleton organization, cell death and translation elongation , pathwayAt the 48 h, 3919 p-sites from 3785 phosphopeptides in 1575 phosphoproteins were identified (3601 p-sites from 3273 phosphopeptides in 1350 phosphoproteins of worker embryos and 2260 p-sites from 1973 phosphopeptides in 971 phosphoproteins of drone embryos). Phosphoproteins in worker embryos belonged to the following functional groups: protein phosphorylation, positive regulation of catalytic activity, chromatin organization, and mRNA processing . In dronBoth castes enriched and shared when comparing the biological pathways between the worker and drone embryos, MAPK signaling pathway, RNA transport, mRNA surveillance pathway, and circadian rhythm . PathwayAdditionally, 18 kinases with high activity were compared in both embryos of worker and drone bees . KinasesThirteen high-activity kinases were selected to compare the difference in site occupancy between worker and drone embryos . The kinAmong the 1575 phosphoproteins in both castes, 75 phosphoproteins altered their expression levels, of which 6 and 69 were up-regulated in worker and drone, respectively . The up-At 72 h of age, 3933 p-sites from 3815 phosphopeptides in 1765 phosphoproteins were identified (3780 p-sites from 3368 phosphopeptides in 1457 phosphoproteins of worker embryos and 1622 p-sites from 1509 phosphopeptides in 836 phosphoproteins of drone embryos). Proteins in worker embryos were involved in protein phosphorylation, regulation of signal transduction, mRNA processing, and cytoskeleton organization . In dronIn comparing the pathway coverage between worker and drone embryos, pathways of RNA transport, mRNA surveillance pathway, MAPK signaling, spliceosome, dorso-ventral axis formation, and circadian rhythm were enriched and shared in both castes . MoreoveFurthermore, 15 kinases with high activity were found in both worker and drone embryos . KinasesAdditionally, the site occupancy of 11 high-activity kinases was compared between worker and drone embryos . GeneralOf the 1765 phosphoproteins expressed by worker and drone embryos at 72 h, 151 were changed in their abundance level . Among t564 in MAPK3 was greater in worker embryos at 24 h. The site abundance of Tyr15 in CDK1 was higher in worker embryos at 48 h.To validate the p-site abundance differences of the crucial kinases between the embryos of worker and drone bees, two p-sites in 2 kinases were selected for Western Blotting analysis . The abuFurthermore, the activity of CDK5 was higher in 48 h worker and drone embryos; enzymatic activity of MAPK3 was higher in drone than worker at 24 h, as well as the activity of CDK1 in 48 h embryos .To verify the function of CAMK2 on the regulation of embryogenesis, RNA interference was tested, simulating a honey bee mutant lacking functional CAMK2. The abundance of the target decreased significantly on the following day, both in haploid and diploid embryos , indicatTo explore the regulatory mechanisms during honey bee embryogenesis, we reported the hitherto most in-depth coverage of phosphoproteome and kinome at time-resolved resolution. There were 6577 p-sites in 6342 phosphopeptides from 2438 phosphoproteins and 168 kinases. In general, embryos across the whole developmental process of both worker and drone bees employ similar phosphoproteome arsenals to regulate embryogenesis. However, distinct phosphoproteome signatures at different phases of embryos have developed in the worker and drone bees to reinforce embryogenesis.via PI3K/AKT or Ras/ERK signaling pathways, contributing to the accumulation of raw organic material , shaping of the germ band and germ layer (26–46 h), and organogenesis (46–72 h). This is in line with specific phosphoproteome signatures that have evolved to match the age-specific physiology . At 24 hmaterial . Moreovematerial , are likmaterial . In 48 hmaterial .259 manifests the fact that the kinase activity is enhanced, which is critical to stimulating cell growth, differentiation, and apoptosis during embryogenesis  catalyzed by PRKCZ/aPKC harms kinase activity   indicateactivity . The obsactivity , validatactivity , indicatactivity , thus leactivity . The hypactivity , relativactivity . These kactivity . Conside2+ signaling pathways, regulating developmental processes such as cytoskeletal rearrangements, dorsoventral patterning, and embryo tissue separation  is negative to the kinase activity (637 in Abl of drone embryos (The last phase of embryogenesis mainly configures the tissues and organs, which is different from the previous stages. As expected, the enriched pathways in worker embryos will likely serve this exact purpose . Example embryos  is close embryos . This in embryos . Its deg embryos . The hig embryos  suggests embryos  implies  embryos . The phoactivity . The obs embryos  may redu embryos . The act embryos . In brieThis study provides a comprehensive resource that reveals phosphorylation-mediated regulatory mechanisms during honey bee embryogenesis. The identified >6000 p-sites, >140 kinases, and the subcellular and chromosome information are the first reported to reveal the phosphorylation networks in honey bee embryos. This offers a valuable resource for signaling research on regulatory sites, kinases, and dynamics in honey bee embryos. Further study is encouraged to understand the role of regulated p-sites and kinases during honey bee embryogenesis.


Mycobacterium tuberculosis complex (MTBC) characterization. Herein, we developed a house-made targeted next-generation sequencing (tNGS) spoligotyping, and compared its outputs with those of membrane-based spoligotyping. A total of 144 clinical MTBC strains were retrospectively selected to be representative of the local epidemiology. Data analysis of a training set allowed for the setting of “presence”/“absence” thresholds for each spacer to maximize the sensibility and specificity related to the membrane-based spoligotyping. The thresholds above, in which the spacer was considered present, were 50 read per millions for spacers 10 and 14, 20,000 for spacers 20, 21, and 31, and 1000 for the other spacers. The confirmation of these thresholds was performed using a validation set. The overall agreement on the training and validation sets was 97.5% and 93.8%, respectively. The discrepancies concerned six strains: Two for spacer 14, two for spacer 31, and two for spacer 32. The tNGS spoligotyping, whose thresholds were finely-tuned during a careful bioinformatics pipeline development process, appears be a technique that is reliable, inexpensive, free of handling errors, and automatable through automatic transfer into the laboratory computer system.Epidemiological studies investigating transmission chains of tuberculosis are undertaken worldwide to tackle its spread. CRISPR locus diversity, called spoligotyping, is a widely used genotyping assay for  Mycobacterium tuberculosis complex (MTBC) transmission in high-income and low-tuberculosis (TB) prevalence countries remains a public health priority considering the constant changes in MTBC epidemiology worldwide. Key measures for TB control rely on the linkage of cases and on the identification of transmission chains, performed through a population-based systematic molecular TB survey to uncover outbreaks, even between apparently unrelated cases [The control of ed cases ,2. ThereThe analysis of clustered regulatory interspaced short palindromic repeats (CRISPR) locus diversity, called spoligotyping, is a widely used genotyping assay for the characterization of MTBC and epidemiological purposes ,4. HistoNevertheless, in silico WGS-based spoligotyping requires large amounts of pure mycobacterial DNA extract. For proximity laboratories unable/unwilling to provide high inoculum MTBC cultures (requiring BSL3 facilities and costly transportation), it was necessary to develop an in silico spoligotyping technique compatible with inactivated samples containing low DNA amounts. A new commercial in vitro diagnostic-targeted NGS test, the Deeplex Myc-TB , allows for the targeting of 18 MTBC drug resistance-associated genes, combined with genomic targets for mycobacterial species identification and MTBC spoligotyping from samples containing low DNA amounts ,12. HoweHerein, we developed and routinely implemented a house-made targeted next-generation sequencing (tNGS) spoligotyping method after a PCR amplification step of the DR locus, feasible from inactivated bacterial lysates containing low DNA amounts. This assay was compared with both the membrane-based method and the in silico WGS-based spoligotyping in terms of output, allowing for a continuity of MTBC molecular surveys at the regional level in a cost-effective manner.Between January 2017 and February 2018, 144 MTBC isolates from specimens sampled from patients during routine care in the Hospices Civils de Lyon, France were retrospectively selected to be representative of the local epidemiology. MTBC strains were separated in two homogenous sets  and the RPM mean value of “absent” spacers was very low  and the RPM mean value of “absent” spacers was the highest  and the RPM mean value of “absent” spacers was low  at the sample level and 99.9%  at the spacer level. The discrepancies concerned two strains, and each time spacer 14 was “absent” with the membrane-based method and “present” with tNGS spoligotyping. For this spacer, the Cohen’s Kappa coefficient indicated that the concordance between membrane-based spoligotyping and tNGS spoligotyping was illustrative of an almost perfect agreement  were also analyzed according to the previously defined thresholds. The overall agreements on the validation set were 93.8%  at the sample level and 99.8%  at the spacer level. The discrepancies concerned four strains: Two strains for spacer 31 that was “present” with the membrane-based method and “absent” with tNGS spoligotyping, and two strains isolated from the same patient for spacer 32 that was “absent” with the membrane-based method and “present” with tNGS spoligotyping. For these spacers, Cohen’s Kappa coefficient indicated that the concordance between membrane-based spoligotyping and tNGS spoligotyping was illustrative of an almost perfect agreement  at the sample level and 99.9%  at the spacer level. Of note, these discrepancies had no impact on the identification of MTBC species or of To investigate the discrepancies between membrane-based spoligotyping and tNGS spoligotyping, in silico spoligotyping using WGS was generated for 144 MTBC strains, allowing for a reconstruction of the CRISPR locus in MTBC using CRISPR-builder TB [Overall, spacer 31 was the spacer related to most of the discordance between in silico spoligotyping and the other methods based on the initial PCR.Concerning the two discrepant MTBC isolates between membrane-based and tNGS spoligotyping of the training set , the in silico spoligotyping was concordant with the tNGS method. The reconstruction of the CRISPR locus using CRISPR-builder TB did not find any genetic element that could explain the discrepancies. Concerning the two discrepant MTBC isolates on spacer 31 between membrane-based and tNGS spoligotyping of the validation set (“present” with the membrane-based method and “absent” with tNGS spoligotyping), the in silico spoligotyping was concordant with the membrane-based method. For these MTBC isolates, CRISPR-builder TB found an insertion of the insertion sequence (IS) 6110 within the DR sequence upstream the spacer 31 for one strain, and within the DR sequence downstream the spacer 31 for the other strain. Concerning the two discrepant MTBC isolates on the spacer 32 between membrane-based and tNGS spoligotyping of the validation set (“absent” with the membrane-based method and “present” with tNGS spoligotyping), the in silico spoligotyping was concordant with the tNGS method. For these MTBC isolates, CRISPR-builder TB found an insertion of the IS6110 within the DR sequence upstream of spacer 32 for both strains.In the present study, the finely-tuned development of the “house-made” bioinformatics pipeline was a key step in the development of tNGS spoligotyping. This method includes an initial PCR step for the amplification of the spacer sequence. In addition, the spacer “presence”/“absence” thresholds should be different from a technique without amplification, e.g., in silico spoligotyping using WGS, to avoid the risk of false positive results related to inter-sample contaminations. Therefore, NGS data analysis using free online pipelines, such as SpoTyping , was impAfter the finely-tuned development of the “house-made” bioinformatics pipeline to define the appropriate detection thresholds for each of the spacers, the tNGS spoligotyping relying on the “house-made” pipeline analysis had a very good overall agreement with the membrane-based method at the sample level. The overall agreement was significantly higher than the one found between the membrane-based method and the in silico spoligotyping using WGS . The recIn terms of reaction cost, tNGS spoligotyping (around EUR 24 per sample) is more low-cost than the membrane-based method (around EUR 30 per sample) or the in silico spoligotyping using WGS (around EUR 28 per sample). Moreover, tNGS spoligotyping has the advantages of requiring less technical staff compared to the membrane-based method, and of being feasible using inactivated bacterial lysates containing few DNA amounts compared to in silico spoligotyping using WGS. The tNGS spoligotyping method could be performed by NGS-equipped national or supranational reference centers in low-income countries, from samples sent by outlying (remote) TB diagnosis centers to ensure a national epidemiological survey. As the diversity of the CRISPR locus has been shown to accurately reflect the phylogeny of MTBC, spoligotyping can be used not only for epidemiological purposes, but also for MTBC species identification using an algorithmic approach in routine TB diagnosis . Consequhttps://github.com/dcouvin/SpolLineage (accessed on 13 March 2021) [Membrane-based spoligotyping experiments were performed as described elsewhere . MTBC spThe first step PCR was directly performed on warm inactivated mycobacterial lysates without preliminary DNA extraction. Briefly, the PCR mix contained the same primers as those used for membrane-based spoligotyping, except for being biotin-free  and a Platinum™ SuperFi™ polymerase . The PCR reaction was performed in a thermal cycler peqSTAR  according to the following program: 1 cycle of DNA denaturation at 98 °C for 30 s; 30 cycles of amplification at 98 °C for 10 s (denaturation); 55 °C for 10 s (hybridization); 72 °C for 60 s (elongation); and 1 cycle at 72 °C for 5 min. The obtained amplicons were diluted twice in PCR-grade water and stored at +4 °C until sequencing.Libraries were generated using a bead-based tagmentation system . A nanoliter liquid handler  was used to reduce the reaction volumes by 10 times. Miniaturized libraries were sequenced on the Nextseq system (Illumina) to produce 150 base-pair paired-end reads. A minimum number of sequenced reads of 100,000 reads was obtained for each isolate.https://gitlab.inria.fr/HCL/software/spomutscan (accessed on 13 March 2021).To ensure a continuity in the interpretation of spoligotyping results, the membrane-based method used for 30 years was considered as the reference method for data analysis and choice of the tNGS threshold. For this purpose, a “house-made” pipeline SpoMutScan was developed. SpoMutScan code leverages MutScan capabilities , coupledA list of 43 strings, corresponding to the 43 spacers, was included in the image. Those strings were prepared to be in the format adapted to MutScan as input. A total of 43 MutScan commands were then generated, one for each spacer. MutScan outputted the raw number of reads containing the targeted strings. SpoMutScan counted and maintained the total number of reads presented as input. Each raw count for the spacers was then normalized against the total number of sequenced reads and multiplied by 1 million . The purpose of this normalization was to set up RPM thresholds independent from the sequencing depth of samples. Eventually, the SpoMutScan generated binary spoligotypes regarding the previously mentioned RPM thresholds (different spacers requiring different thresholds). From binary spoligotypes, octal spoligotypes were derived. SpoMutScan outputted two files: A log file containing normalized counts for each spacer, and a file with binary and octal spoligotypes.For MTBC WGS, genomic DNA was purified from cleared lysates using the Maxwell RSC Instrument  automated DNA extraction system and the Maxwell RSC Blood DNA Kit (Promega). Libraries were generated using a bead-based tagmentation system . A nanoliter liquid handler  was used to reduce the reaction volumes by 10 times. Miniaturized libraries were sequenced on the Nextseq or Miseq system (Illumina) to produce 150 or 300 base-pair paired-end reads, respectively. Reference genome coverage was at least 96% and the depth of coverage at least 30×.https://github.com/xiaeryu/SpoTyping-v2.0; accessed on 13 March 2021) [MTBC in silico spoligotypes were determined through the open access tool SpoTyping (ch 2021) .https://github.com/cguyeux/CRISPRbuilder-TB, accessed on 13 March 2021) [To identify the potential events responsible for discrepancies between membrane-based and tNGS spoligotyping , MTBC WGS was performed and allowed for the attainment of in silico spoligotyping and the reconstruction of CRISPR locus using CRISPR-builder TB (ch 2021) .Violin plots, receiver operating characteristic (ROC) curves, and plots of sensibility/specificity according to the thresholds were built using R (R Core Team (2018). R is a language and environment for statistical computing . Cohen’s Kappa values were calculated using XLSTAT 2020.5.1  and interpreted according to Landis and Koch criteria .Hospices Civils de Lyon since April 2022.In conclusion, tNGS spoligotyping is a reliable spoligotyping method, feasible from inactivated bacterial lysates containing low DNA amounts, free of handling errors, automatable through automatic transfer into the laboratory computer system, and allow for a continuity of MTBC molecular surveys at the regional level in a cost-effective manner. Therefore, this technique has been implemented as a routine in the mycobacterial laboratory of the


The volume of the analyzed wastewater reflected the active sludge load in the real biological treatment system. This research was carried out by the method of short-term tests. The nitrification process was inhibited to the greatest extent by wastewater from the production of acrylonitrile (approx. 51%) and styrene–butadiene (approx. 60%) rubbers. In these wastewaters, nitrification inhibition occurred due to the high COD load and the presence of inhibitors. Four-fold dilution of the samples resulted in a two-fold reduction in the inhibition of nitrification. On the other hand, in the wastewater from the production of emulsifiers and polyvinyl acetate, a two-fold reduction in COD  resulted in a significant decrease in nitrification inhibition. Wastewater from the production of styrene, solvents  and owipian® inhibited nitrification under the influence of strong inhibitors. Lowering the COD value of these wastewaters did not significantly reduce the inhibition of nitrification.COD is an arbitrary indicator of the content of organic and inorganic compounds in wastewater. The aim of this research was to determine the effect of COD of industrial wastewater on the nitrification process. This research covered wastewater from acrylonitrile and styrene–butadiene rubbers, emulsifiers, polyvinyl acetate, styrene, solvents  and owipian Municipal and industrial wastewater is a carrier of many dangerous organisms and substances, which include parasites and pathogenic organisms ,2,3, toxNitrosomonas, Nitrosovibrio, Nitrosococcus, Nitrosospira and Nitrosolobus transform ammonium nitrogen to nitrite ;2-N + NO3-N) in the flask with the tested sewage, after 4 h of aeration [mg·dm−3];2-N + NO3-N) in a flask with a comparative inhibitor (ATU) after 4 h of aeration [mg·dm−3].The volume of the mixture in each flask was 250 cmAhlstrom Munktell 389 filters were used to filter the samples.®. Wastewater in the amount of 25 cm3, 50 cm3, 75 cm3 and 100 cm3 was used for the tests, with a variable COD value.The study used wastewater inhibiting the nitrification process to a degree higher than 30.0%, coming from the production of styrene–butadiene and acrylonitrile rubbers, styrene, solvents , emulsifiers, polyvinyl acetate and owipian4-N, NO2-N and NO3-N were determined by the colorimetric method using the CADAS 30S biochemical spectrophotometer  and appropriate cuvette tests. Measurements were made in triplicate.The concentrations of NH3-N and NO2-N) in the control flask and the flask with the comparative inhibitor, after 4 h of aeration, using Formula (2):−1];2-N + NO3-N) in the control flask after 4 h of aeration [mg·dm−3];2-N + NO3-N) in a flask with a comparative inhibitor (ATU), after 4 h of aeration [mg·dm−3];−3].The nitrifying activity of the activated sludge was calculated based on the differences in the concentration of oxidized forms of nitrogen :−1];3];−3];−3].The specific rate of nitrification was determined on the basis of changes in NH−3, and the values of the nitrification inhibition parameter (IN), expressed in%, were analyzed. The normality of individual data distributions was tested with the Shapiro–Wilk test. In the null hypothesis H0 for this test, it was assumed that the given research sample comes from a normally distributed population. The significance level for this test was assumed to be α = 0.05. The results of the Shapiro–Wilk test for measurement data, including COD and IN values, are presented in Before the statistical analysis was performed, the measurement data were verified with regard to outliers. In order to apply appropriate parametric or nonparametric tests of outlier detection, at the beginning, the statistical distributions of individual observational data strings were assessed. The COD values, expressed in mg·dmp-value, there is no reason to reject the null hypothesis that the distribution of individual data is normal. The normality of the distribution of measurement data allowed for the verification of outliers by applying the parametric Grubbs’ T-test. The calculations were performed with the statistical package Statistica v. 13.1 . The results of the Grubbs’ T-test are summarized in Since for each data string, the assumed significance level α = 0.05 is lower than the calculated value of the test probability 0 stating that there are no outliers in a given data sequence.The data in 2 dm−3, while the dependent variable was the parameter IN, given as a percentage, calculated according to Formula (1). The relationship between the tested parameters in individual types of industrial wastewater is described by Equation (4):Statistical analysis of linear regression was used to determine the strength and direction of the impact of the COD value in industrial wastewater on the process of nitrification inhibition. The independent variable in this analysis was the value of COD in the wastewater, defined in mgO2 were verified by the Fisher–Snedecor F-test. Statistical calculations, along with the verification and estimation of the parameters of the regression equation, were performed with the Statistica v. 13.1 computer program .The parameters of the linear regression model were determined by the least-squares method. The significance of the relationship between the analyzed variables, the significance of the slope coefficient of the regression equation and the significance of the coefficient of determination RIn studies conducted on wastewater from acrylonitrile and styrene–butadiene rubber production, the inhibition of nitrification occurred due to the concentration of pollutants  contained in both wastewater samples and the presence of inhibitors. In the wastewater from the production of acrylonitrile rubbers, a two-fold reduction in the COD value resulted in a reduction in nitrification inhibition from 50.7% in the initial sample to 19.8%. As the wastewater was diluted further, the inhibitory effect was further reduced .2·dm−3 caused only a two-fold reduction in nitrification inhibition (from 59.8% to 28.6%). Therefore, it can be concluded that reducing the concentration of pollutants in the tested sample to the COD value of 156.8 mgO2·dm−3 does not satisfactorily reduce the inhibition observed in the sample with the maximum concentration of pollutants  compared to the initial sample resulted in a decrease in nitrification inhibition from 44.5% to 17.0%  resulted in a decrease in nitrification inhibition from 38.9% to 16.1% . In the case of sewage from styrene production, the inhibition of nitrification in the initial sample was slightly lower , but after four-fold dilution, it remained at a relatively high level, 37.5%, with a COD of only 60.5 mgO2·dm−3  and owipiangO2·dm−3 .2·dm−3 to 122.9 mgO2·dm−3, did not remove the inhibition effect. The degree of inhibition of the nitrification process remained at the level of approx. 50.0%; thus, it was comparable to the initial sample  production, despite the decrease in COD from 491.6 mgOl sample .® production. The reduction in the concentration of organic pollutants in the form of COD from 621.3 mgO2·dm−3 to 155.3 mgO2·dm−3 did not reduce the inhibition of nitrification, which still amounted to approx. 50.0%  and owipian®. In this case, it is likely the factors limiting nitrification were the inhibitors present in the tested chemical sewage. It is also possible that this effect was increased by numerous disinfecting compounds present in municipal wastewater, used during COVID-19 [The constantly developing chemical industry affects the degradation of the natural environment, the protection of which requires large financial outlays related to the necessity of neutralizing the resulting wastewater. Pretreated wastewater from production plants is usually directed to a mechanical and biological wastewater treatment plant . DespiteCOVID-19 .According to the literature data, the amount of nitrogen removed from the system in the form of biomass ranges from 20 to 30% . These dSignificant factors limiting or even preventing the course of nitrification in the biological treatment system of the Municipal and Industrial Sewage Treatment Plant in Oświęcim are inhibitors flowing in with industrial sewage and high COD values. The list of organic and inorganic compounds having a negative effect on nitrifying bacteria is very long and includes a number of substances for which the degree of inhibition has been determined ,17,19,20® production. In the tested sewage, sometimes even a four-fold reduction in the concentration of organic pollutants, expressed in the form of COD, did not satisfactorily eliminate the inhibition process. Similar observations were made by Samudro et al. [As part of this study, the degree of nitrification inhibition under the influence of various concentrations of chemical pollutants  present in selected industrial wastewater was assessed. The research was carried out to determine the COD value in postproduction wastewater, which inhibited the nitrification process. The obtained results clearly show that the high concentration of organic pollutants in most of the tested wastewater was one of the main reasons for a significant reduction in nitrification. This is evidenced by the results obtained during the testing of wastewater from acrylonitrile and styrene–butadiene rubbers, styrene, solvents  and owipiano et al. , who exaThe conducted research showed that in the biological wastewater treatment system at the Municipal and Industrial Wastewater Treatment Plant in Oświęcim, the nitrification process is clearly disturbed by high COD values and the presence of inhibitory substances in the incoming wastewater.The diversified production profile in the chemical industry hinders the proper operation of mechanical and biological wastewater treatment plants. In such plants, the amount and composition of sewage as well as the concentration of organic pollutants expressed in the COD value are subject to continuous fluctuations ,39. The ®. The main reason for the low efficiency of the nitrification process at the WWTP in Oświęcim is the concentration of organic pollutants present in the sewage and the presence of inhibitors. COD values exceeding 400 mgO2·dm−3 caused nitrification inhibition above 50%. COD values below 200 mgO2·dm−3 only slightly reduced the efficiency of this process, with the exception of sewage from the production of styrene, solvents  and owipian®. Diluting industrial wastewater with municipal wastewater at a ratio of 1:3, or even 1:1, has a positive effect on the treatment processes, but still does not guarantee the elimination of nitrification inhibition. At present, industrial wastewater destined for biological treatment is mixed with municipal wastewater in a proportion of 2:1 or even 3:1, and it is not possible to dilute it further, due to the insufficient volume of available municipal wastewater. Only a clear advantage of typical domestic sewage in the system would allow for a partial reduction in nitrification inhibition. Nitrifying bacteria present in the activated sludge of the WWTP in Oświęcim are subject to the phenomenon of chronic stress. This is evidenced by the lower nitrification activity of activated sludge than in the literature, which in the period studied was on average about 1.0 mg·g·h−1 of oxidized nitrogen. It should be emphasized, however, that under favorable conditions, this activity is sufficient to maintain the parameters of the water-legal permit for the removal of ammoniacal nitrogen from wastewater.Industrial wastewater contains numerous chemicals, including toxic ones, which significantly contribute to the inhibition of the nitrification process. In the Municipal and Industrial Wastewater Treatment Plant in Oświęcim, the most dangerous industrial wastewater comes from the production of acrylonitrile and styrene–butadiene rubbers, emulsifiers, polyvinyl acetate, styrene, solvents  and owipian


It was found that distortion healing abutments screws were seen after 32 times screwing and unscrewing. There was a significant difference in the distortion  after 24 times of repeated usage of healing abutment and at 160, 320, and 400 times. No surface distortions were observed at the healing abutment screw head at 4, 8, 16, 24, 32, 40, 80, 160, 320, and 400 cycles of screwing-unscrewing. It can be concluded that repeated screwing and unscrewing of the implant-healing abutments causes damage to the healing abutment hex surface. The distortion of healing abutments screws was seen after 32 times screwing and unscrewing. No surface distortions were observed on the healing abutment screw head until 400 times of screwing and unscrewing. Hence, the clinician should be cautious while using the healing abutments repeatedly.Little has been studied regarding the repeated usage of healing abutments and their effects on the distortion of dental implant-healing abutment hex surfaces. Additionally, implant manufacturers do not provide specific guidelines on how many times a healing abutment can be used before discarding. Therefore, we evaluated the effect of repeated screwing-unscrewing of implant-healing abutment on the healing abutment hex surface and screw head. A total of 12 Biomate implants with 4 mm diameter and 13 mm length were inserted into a synthetic bone block. The standard healing abutments of 3 mm diameter and 4 mm length were screwed onto each implant using a torque ratchet at a final torque of 30 Ncm. Immediately, the abutments were unscrewed at 30 Ncm. Then, screwing-unscrewing was repeated for 4, 8, 16, 24, 32, 40, 80, 160, 320, and 400 times and the healing abutments were scanned under the scanning electron microscope for any distortion. Distortion was graded as 0, 1, 2, and 3. Data were analyzed using SPSS 24.0. Descriptive statistics were calculated. One-way ANOVA with post hoc using Tukey’s HSD test was performed to analyze the difference in distortion at different screwing-unscrewing times. A significant level was selected at  Dental implants with digital technologies are widely used for the prosthetic rehabilitation of a missing tooth . A typicHealing abutments are temporary components placed on the implant body during the healing period and aid in providing healthy peri-implant mucosa to support the future tooth prosthesis. Implant components are usually manufactured from titanium or its alloys, although newer materials are being tried . The mecThe screw design of the healing abutment and its physical properties vary greatly between manufacturers. The differences in screw design and physical property among healing abutments of different manufacturers may be attributed to the external diameter, thread depth, precision of taper, components, and poor tooling of the healing abutment. These may cause significant differences in complications especially screw loosening ,7.Poor connection between healing abutment screw and implant internal hex connection may cause implant-related complications, such as implant-crown dislodgement. Although the interface between the healing abutment screw and implant internal hex connection is important, no studies have been carried out on the biomechanical integrity of the healing abutment screw and implant internal hex connection. Hence, we evaluated the distortion of hex threads of a used healing abutment microscopically .Repeated use of healing abutment may lead to plastic deformation due to repetitive preloads during tightening and retightening. This plastic deformation might cause screw loosening and accidental removal of the abutment in the patient’s oral cavity or might cause micro gaps between the implant-abutment interface . The perDecontamination of implant parts, especially used healing abutments, is important and is achievable through procedures such as autoclave, NOC, air polishing, electrolysis, etc. ,14,15. AThe reusability of healing abutments has often been considered debatable owing to the lack of universal consensus or guidelines. In addition, the implant manufacturers have not provided any recommendations for the repeated usage of their healing abutments, although there is an effect of screwing-unscrewing of healing abutment on the healing abutment hex surface and screw head ,17. HencThis study used dental implants and healing abutments, bone block, dental implant torque ratchet , and SEM . The characteristics of the dental implant parts are summarized in In this study, 12 Biomate implants were used. The sample size calculation was conducted following previous studies in which the sample size ranged from 10 to 15 ,22. ImplThe standard healing abutment (HA) of 3 mm diameter and 4 mm length was used in our study. The 12 healing abutments were divided into two groups; the first group (HA 1 to HA 6) was screwed-unscrewed 4, 16, 32, 80, and 320 times . The secOne-sided grooves were created on the healing abutment by an ai-rotor handpiece using straight diamond bur for placement, orientation, and identification during the microscopic procedure. Each healing abutment was sent for the SEM evaluation after the aforementioned times of screwing and unscrewing. Two parts of the healing abutments were evaluated under the SEM; first, the connecting screw head of the healing abutment, and second, the screw thread of the healing abutments. The external surface of the healing abutment screw consisting of thread for connection to the corresponding internal hex of the dental implant was hypothesized to deform after repeated loading and unloading. The hollow portion over the superior surface of the screw wherein the screwdriver gets embedded to tighten or loosen the screw from the implant .All the 12 healing abutments were examined using the SEM after repeated screwing and unscrewing. The healing abutments were again scanned after 4, 8, 16, 24, 32, 40, 80, 160, 320, and 400 insertion and removal cycles. Abutment screws were positioned on separate SEM pin-type mounts using conductive adhesive carbon tabs.The healing abutments were gold-sputtered prior to SEM procedures to make the samples more electroconductive . The hexThe surface distortion of the implant-healing abutment screw (hex thread) was determined under the SEM by using the following criteria using an ordinal scale  27,28].,28.27,28Grade 0: Thread surface intact with no visible surface irregularity.Grade 1: Mild surface irregularities with pitting or slight cracking on the thread surface.Grade 2: Moderate surface irregularities with deformed thread crests with pitting and slight cracking on the thread surface.Grade 3: Severe surface irregularities with advanced deformation of thread crests.The implant-healing abutment screw head distortion was determined under SEM were determined by using the following criteria using an ordinal scale  27,28].,28.27,28Grade 0: Screw head intact with no visible surface irregularity.Grade 1: Mild surface irregularities with pitting or slight cracking on the screw head.Grade 2: Moderate surface irregularities with deformed screw head with pitting and slight cracking.Grade 3: Severe surface irregularities with advanced deformation of the screw head.p-value = 0.05. Only one researcher performed all the research procedures. Then, in the end, all data were verified by random measurements, and data were found valid.Data were analyzed using SPSS 24.0 . Descriptive statistics were calculated. One-way ANOVA with post hoc using Tukey’s HSD test was performed to analyze the difference in distortion at different screwing-unscrewing times. Significance level was selected at No surface distortions occurred at the healing abutment screw threads up to 16 cycles of screwing-unscrewing . HoweverThe descriptive statistics of surface distortions of the healing abutments after the specific cycles of screwing-unscrewing are shown in p-value < 0.05) after 24 times of repeated usage of the healing abutment.Multiple comparisons  show thaNo surface distortions were observed at the healing abutment screw head at 4, 8, 16, 24, 32, 40, 80, 160, 320, and 400 cycles of screwing-unscrewing .We aimed to examine if repetitive screwing and unscrewing could affect the surface characteristics of the healing abutment. This is the first study to investigate the same to the best of our knowledge. The null hypothesis is rejected, and there are some surface changes following the screwing-unscrewing of healing abutment on the implant hex surface and screw head. Various methods to measure the surface wear are the 2D surface profilometry ,30, SEM From this study, the microscopic pictographs confirmed this at higher resolution taken after recurring cycles, in which we observed thread and hex driver insertion surface distortion of healing abutments. We also found that the abutment removal torque gradually lowed than the insertion torque with increased repetitive screw unscrew cycles. These results could be explained based on the behavior of prosthesis abutment and hence would be discussed accordingly henceforth.The friction helps in the retention of the restoration, contributes the joint stability, and resists screw loosening. Increasing screw insertion cycles decreases the friction between the screw and internal threads of the implant. We observed that after 24, 32, and 40 times repeated cycling of loosening and tightening of healing abutment causes mild surface irregularities with pitting or slight cracking on the thread surface of healing abutment occurring in all the tested healing abutments. This has been confirmed by SEM analysis that increasing screw closure cycles caused changes in the surface of the screw. The clinical implications of the screwing and unscrewing are shown in Our findings were in concordance with the results of Delben et al. , CarsdosAnother factor responsible could be the brittleness of titanium screws due to the body-centric cubic crystal structure making them more vulnerable to distortion . Our finAssunção et al.  studied The coefficient of friction among two metals is maintained by their intrinsic metallurgic properties and the mechanized process, which determines their geometric design and surface texture, which might be the reason previous studies observed differences in pre-facture torque value of abutments from different brands and within the same brand, and different shipments as well ,25.In a clinical setting, surface distortion could lead to the formation of micro gaps, causing microleakage of bacterial substrates or accidental slippage of the abutment in the oral cavity, which might lead to its aspiration ,40.The mechanical properties of the implant and the screwdriver  can affeThis study can be extended to study the wear by advanced imaging methods in the future. In addition, the type of stress distribution can be studied using the FE study. The in vitro study design of the present study is one of its potential limitations as it could not simulate the exact biologic surroundings of the oral cavity. Thus, the findings should be validated in a clinical condition. The decontamination (autoclaving) of the healing abutments is performed in between repeated use , but we Within the limitation of this study, it can be concluded that repeated screwing and unscrewing of the implant-healing abutments causes damage to the healing abutment hex surface. The distortion of healing abutments screws was seen after 32 times screwing and unscrewing. No surface distortions were observed on the healing abutment screw head until 400 times of screwing and unscrewing. Hence, the clinician should be cautious while using the healing abutments repeatedly.


The paper presents a comparative analysis of two extrusion methods of biocomposites with a poly (PHBV) matrix filled with flax and hemp fibers in terms of biopolymer production, its processing in the further injection process, and an evaluation of the mechanical and functional properties of the products. Biocomposites containing 15% by weight of the filler were produced using single- and twin-screw extruders. The biocomposites were then processed by injection molding and then, among other things, the pressures in the mold cavity during processing were analyzed. The produced samples were tested by means of the following tests: uniaxial tensile strength, hardness, and impact tensile strength. The biocomposite’s microstructure was also analyzed using scanning electron microscopy (SEM), as were the shrinkage and water absorption of the manufactured products. In addition, thermal gravimetric analysis (TGA) and differential scanning calorimetry (DSC) tests were performed. It was found that the extrusion method changed significantly the geometry of the filler fibers and the processing capabilities of the manufactured materials. Significant differences in the mechanical and functional properties of the obtained biocomposite products were also found. On their basis, the advantages and disadvantages of both extrusion methods were discussed. Most of the obtained properties of injection products indicate the choice of single-screw extrusion. The products were characterized by slightly better mechanical properties and lower processing shrinkage. In turn, composites obtained by the screw method were characterized by lower water absorption and lower viscosity of the composite during injection molding. New polymeric materials with increasingly improved properties and lower production costs are still being sought. The growing ecological awareness of societies leads to the search for environmentally friendly materials with such characteristics as biodegradability, natural origin, and renewability. The materials are becoming more and more important. This shapes the direction of production and an improvement of a new type of polymeric materials, from non-renewable, difficult to degrade, or completely non-biodegradable polymers to renewable ones of natural origin which are fully biodegradable ,2,3. TheCellulose fibers as a filler in a polymer matrix are characterized by low production costs compared to synthetic fibers, and they also have appropriate strength properties that can be a great alternative to synthetic fillers ,5. BiocoGreen polymers utilize beneficial features such as an ability to produce from renewable raw materials and biodegradation, and are unique and most desirable polymer material in the current problem of waste management. One of the interesting materials of this type are polyhydroxyalkanoates (PHA), which are characterized by biocompatibility, non-toxicity, and the fact that they are subject to enzymatic degradation. The main polymers from the PHA group include poly(3-hydroxybutyric acid) (PHB), which is produced in cells of Ralstonia eutropha bacteria as a reserve material ,13,14,15Positive features of cellulose fillers, such as low density at maintaining high strength and stiffness, lower production costs, renewability, and no problems with recycling, make it possible to effectively use them as fillers in the polymer matrix. It should be noted, however, that fibers of plant origin are characterized by a different chemical composition, including the share of cellulose, hemicellulose, and lignin, which directly affects their properties ,20. In oPHBV, under the trade name Enmat Y1000 of Helian Polymers , in powder form, was used as the polymer matrix. Flax and hemp fibers produced by EKOTEX  with a length of 1 mm were used as a filler in the polymer matrix. The mass fraction of fibers in the polymer matrix was 15 wt.%. Flax fibers were alkalized with 2% NaOH solution, while hemp fiber was alkalized with a 10% NaOH solution. The concentrations of the solutions were selected based on the results of the tests described in .The biocomposites were produced on a twin-screw extruder (co-rotating method) and a single-screw extruder.H15TWIN—biocomposite with hemp fiber, produced using a twin-screw extruder;H15SINGLE—biocomposite with hemp fiber, produced using a single-screw extruder;F15TWIN—biocomposite with flax fiber, produced using a twin-screw extruder;F15SINGLE—biocomposite with flax fiber, produced using a single-screw extruder.Due to the better presentation of the results, a set of markings used in the further part of the work was developed:PHBV biocomposites filled with a fiber of plant origin were extruded using a ZAMAK REA-2P12A Explorer twin-screw extruder  . The tecPHBV biocomposites filled with the fiber of plant origin were also extruded using a ZAMAK EHP-25E single-screw extruder  . TechnicInitially, an attempt was made to extrude PHBV biocomposites with similar processing parameters as in the case of the twin-screw method. For the given similar values of temperatures of individual heating zones of the single-screw extruder, a high increase in extrudate viscosity was observed, whose consistency visually resembled a solid with a tendency to brittleness. Therefore, an attempt was made to optimize the processing parameters in order to obtain an extrudate with similar rheological parameters  as produced using a twin-screw extruder. For this purpose, the temperature of the extruder head was increased to 170 °C  and the For the production of samples in the injection molding process, a DrBoy 55E injection molding machine  equipped with the Priamus system was used to monitor and control the injection molding process. An injection mold with inserts for uniaxial tensile testing (according to EN ISO 527-1) was used for the tests. All four types of biocomposites were injected with the same adjustable parameters presented in The analysis of the pressure profile in the cavity of the injection mold  for biocA TA Instruments DSC Q2000 differential-scanning calorimeter  was used for the thermal tests. All analyses were performed under a nitrogen atmosphere with a relatively constant flow of approximately 50 mL/min.In order to assess the degradation temperature of the obtained biocomposites, TGA  tests using the Mettler Toledo  TGA device were carried out. In this method, the change in the mass of the tested sample with respect to the increasing temperature was determined. The samples were heated to 700 °C at a rate of 10 °C/min. Weight loss ranging from 1 to 10% was monitored.M), and relative elongation at maximum tensile stress (εM). The results were evaluated through the statistical analysis. The arithmetic mean  uniaxial tensile testing machine was used to determine the strength properties of the obtained composites. The uniaxial tensile test was conducted in accordance with the EN ISO 527-1 standard for molded pieces with the dog-bone shape. Each series of samples consisted of seven pieces. Based on the obtained test results, the followings were determined: Young’s modulus (E), tensile strength  and in the gripping part (zone B). A Zwick 3106 hardness tester  was used for this purpose. Seven samples were tested in each series.Biocomposite samples were also tested in the impact tensile test. The impact tensile strength was determined in accordance with the PN-EN ISO 8256 standard. The CAEST 9050  pendulum hammer was used for this purpose.The morphology of the samples subject to tensile tests was examined using a HITACHI S-3400 scanning electron microscope (SEM) .The degree of water absorption of the produced samples was tested on the basis of the EN ISO 62 standard. The shrinkage of the molded parts was carried out on the basis of EN ISO 294-4 standard. The primary shrinkage was tested after approximately 3 h, and the secondary shrinkage after approximately 14 days from the production of molded pieces in the injection molding process.Preliminary fiber length measurements were also made using a Nikon LV-100D optical microscope  and E-MAX software. The length and diameter of the fibers were measured on the surface layer of molded pieces for biocomposites extruded using the single- and twin-screw method. For each sample, the measurements were carried out in the same area, in the middle part of the dog-bone sample, intended for the uniaxial tensile test.p, glass transition temperature—Tg, melting temperature—Tm, and crystallization temperature—Tc.When analyzing the results of tests carried out using differential scanning calorimetry (DSC) , slightlFor biocomposites with a mass content of hemp fibers of 15 wt.% and produced by two different extrusion methods, thermal gravimetric analyses were performed. Higher degradation temperatures of biocomposites with flax fibers in relation to composites with hemp fibers were observed . In turnThe obtained samples were also subject to a uniaxial tensile test. Exemplary stress-strain curves are presented in 2. In turn, in the area B, i.e., the gripping part of the dog-bone shaped sample, the hardness values ranged from 78 to 85 N/mm2.In the case of hardness tests , no signWhen analyzing the results regarding the impact tensile strength , it can In the case of secondary shrinkage tests , lower lBased on the results regarding water absorption , a higheMeasurements of the fiber geometry on the surface layer of the compacts in By analyzing the SEM micrographs  of fractThe extrusion of composites with fiber fillers consists of plasticizing the polymer, mixing it with fiber, and then transporting the mixture through the head to produce an extrudate. This process allows better homogenization of the material, which, if produced, (for instance) in the form of granules, will show much better homogeneity. As a consequence, this may result in the better quality of items produced by injection molding. The parameters that affect the properties of composites in the extrusion process include the rotational speed geometry of the screw/screws, number of heating zones, and temperature .The rotational speed of the screw, when extruding composites, should be as high as possible to minimize the residence time of the material in the plasticizing unit, and to maximize the volumetric flow rate. However, when the extrudate is temperature sensitive, such as in cellulosic fibers, the speed of the extruder screw is limited by the maximum shear rate, which can degrade the fibers. Another reason for limiting the rotational speed of the screw is to minimize the degree of presence of air in the plasticizing unit. If the polymer is extruded at too-high speeds, the air may be “trapped”, which causes the formation of small air bubbles in the extrudate . An impoAn important factor related to the temperature of the process is the residence time of the material in the plasticization unit, which may be too short to plasticize the material, or too long, which may lead to material degradation due to overheating ,32. In aAnother important issue is the geometry of the screw in the plasticizing unit of extruders, which varies in individual zones. These zones have different purposes . Shearing can be increased by using mixing and crushing elements . In the The extrusion with the use of single- and twin-screw extruders provides a different course of shearing and homogenization of the processed material during the extrusion process . BasicalThe results of tests produced by single- and twin-screw extrusion of PHBV biocomposites indicate a significant impact of the extrusion method used on their properties. Biocomposites produced using a single-screw extruder were characterized by better mechanical properties in the uniaxial tensile test. This may be due to the fact that the fibers did not undergo mechanical degradation (shortening) during extrusion in this method (compared to biocomposites produced by twin-screw extrusion). The biocomposite produced by single-screw extrusion spent less time in the plasticizing system due to the standard profile of the screw, the larger diameter of the cylinder channel and the screw, as well as the higher rotational speed of the screw. Worse mechanical properties of biocomposites produced by twin-screw extrusion could result from the small diameter of the screw, the presence of grinding zones in the screw profile (which could result in shortening of the fibers), and the large ratio of the screw length to its diameter . It should be mentioned, however, that the biocomposites produced using a twin-screw extruder were characterized by lower porosity and a lower degree of water absorption, which may be due to the fact that the twin-screw extruder was equipped with a degassing module for the plasticized biocomposite during the extrusion process.The phenomenon of fiber shortening may be confirmed by, for example, the observed changes in processing shrinkage in the length direction of the compact. Both for biocomposites with the flax and hemp fibers produced by the single-screw method, the shrinkage along the length was lower than for biocomposites produced by the single-screw method. The fibers act as a kind of reinforcement, where when they are arranged in the longitudinal direction, they limit shrinkage in this direction. Shorter fibers of smaller length and diameter limit this type of shrinkage to a lesser extent, which was observed for biocomposites produced by the twin-screw method.Measurements of the fiber geometry on the surface layer of the molded piece carried out for the composite obtained using the single-screw method indicate the presence of much longer fibers compared to the fibers on the surface of samples obtained from biocomposites produced on a twin-screw extruder, which are less visible. It seems that this may be due to the fact that the fibers in the composite obtained in the single-screw extrusion technology can be much thicker, stiffer, and longer. Due to this fact, they also become more visible on the surface layer. In addition, a reduction in the thickness and length of fibers embedded in a polymer matrix for biocomposites produced by the twin-screw method was also observed, as confirmed by SEM images, also with regard to SEM images of fibers not embedded in the polymer matrix .The observed phenomenon of fiber shortening in the extrusion process was also noted by other researchers. In the work of Baiardo et al. , an extrWhen choosing the right method of extrusion of PHBV-flax fiber biocomposites, the aspect of process parameters and efficiency should also be analyzed. In the case of the production of composites using a single-screw extruder, the temperature of individual heating zones of the extruder had to be increased in order to obtain an extrudate with a similar viscosity as in the case of production using a twin-screw extruder. In the case of using a single-screw extruder, it was possible to set higher rotational speeds, thanks to which the extrusion efficiency was significantly higher. In addition, the availability of single-screw extruders in manufacturing companies is much greater than in the case of twin-screw equipment. On the other hand, the most alarming aspect was that the homogenization of the fiber-matrix phases did not differ significantly in relation to both extrusion methods. In the case of fibers after the twin-screw method, they are shorter, which increases their number in the matrix, hence SEM images can show a greater number of them in the polymer matrix, and thus a denser arrangement. In the case of SEM images for composites produced by the single-screw method, the fibers are larger (they have not been shortened), and thus they are less often embedded in the polymer matrix. By analyzing the results of all tests carried out in statistical terms, it was found that the standard deviation for test samples of individual composites differs slightly with a slight advantage of the twin-screw method, which is an obvious phenomenon. If in the case of any of the methods the reproducibility of the results decreased (the dispersion would be greater), then our assumptions would be directed to the low homogenization of the fiber-matrix phases due to the uneven distribution of fibers in the polymer matrix.The consequences of using a given extrusion method in relation to the subsequent history of composites processing should also be analyzed. In the injection process, higher values of pressure in the molding cavity were noted for biocomposites produced using a single-screw extruder, which may indicate higher flow resistance for biocomposites with fibers that were less shortened or stratified during single-screw extrusion. In this approach, the biocomposite produced by the twin-screw method with fibers of a smaller diameter and length is easier to process in the context of the possibility of filling the mold cavity.Based on the results of the TGA tests, higher degradation temperatures were found in relation to the percentage weight loss for biocomposites produced by the single-screw method. On the other hand, on the basis of DSC studies, slightly higher values of glass transition, melting, and crystallization temperatures were noted for biocomposites produced by the single-screw method. This may be related to the smaller heating surface of the fibers with a greater length and diameter in relation to the chipped fibers of biocomposites produced by the twin-screw method. In addition, changes in the properties of composites may also be the result of polymer degradation, which would explain the changes in transition and melting temperature and the results obtained with TGA. The heating time of the fibers should also be mentioned. In the work of Summerscales et al. , it was A comparative analysis concerning the selection of the extrusion method for biocomposites with a PHBV matrix filled with flax and hemp fibers was performed in this study. In the manufacturing of biocomposites it was not possible to select the same setting parameters of the process due to the different microstructure forms of the obtained extrudate. In order to unify the form of the obtained biocomposites, it was necessary to increase the temperature and speed of extrusion in the single-screw method. This resulted in an increase in extrusion efficiency.In the context of assessing the mechanical properties and quality of the moldings, the deterioration of properties in the uniaxial tensile test and the increase in shrinkage of molded piece for biocomposites produced by the twin-screw method were noted. The reason for this phenomenon is most likely the fact that the fibers during processing with this method were shortened and stratified, and could undergo thermal degradation due to the specific arrangement of the screw zones  and the too-long stay in the plasticization system due to the very high length-to-diameter ratio of the screw.Despite the indicated disadvantages of the twin-screw method, some advantages for the processing of the analyzed biocomposites can be indicated. The fibers are more fragmented, hence lower pressure values in the injection mold were obtained due to lower flow resistance. This allows the easier shaping of products in the injection molding process. In addition, biocomposites produced using a twin-screw extruder were characterized by lower porosity and a lower degree of water absorption, which could be the result of the use of a degassing zone in the twin-screw extruder.Summing up, it is difficult to unequivocally recommend the method of extrusion for biocomposites with PHBV matrix filled with flax and hemp fibers. However, when analyzing the issue of commercialization of this type of biocomposites, the availability of single-screw extruders is greater. The obtained properties of the injection products indicate the choice of single-screw extrusion. The products can be then characterized by slightly better mechanical properties and lower processing shrinkage.