The emergence of SARS-CoV-2 variants is cause for concern, because these may become resistant to current vaccines and antiviral drugs in development. Current drugs target viral proteins, resulting in a critical need for RNA-targeted nanomedicines. To address this, a comparative analysis of SARS-CoV-2 variants was performed. Several highly conserved sites were identified, of which the most noteworthy is a partial homopurine palindrome site with >99% conservation within the coding region. This sequence was compared among recently emerged, highly infectious SARS-CoV-2 variants. Conservation of the site was maintained among these emerging variants, further contributing to its potential as a regulatory target site for SARS-CoV-2. RNAfold was used to predict the structures of the highly conserved sites, with some resulting structures being common among coronaviridae. An RNA-level regulatory map of the conserved regions of SARS-CoV-2 was produced based on the predicted structures, with each representing potential target sites for antisense oligonucleotides, triplex-forming oligomers, and aptamers. Additionally, homopurine/homopyrimidine sequences within the viral genome were identified. These sequences also demonstrate appropriate target sites for antisense oligonucleotides and triplex-forming oligonucleotides. An experimental strategy to investigate these is summarized along with potential nanoparticle types for delivery, and the advantages and disadvantages of each are discussed. The COVID-19 pandemic has caused a global public health crisis. Although current vaccines can be greater than 90% effective at preventing severe disease, there is still grave concern over the emergence of variants which may circumvent currently available vaccines and antiviral drugs still in the research and development phases. Recently, antisense, aptamer, and RNA-based drugs have been clinically approved, with promising preclinical data on the safety of antisense oligonucleotide (ASO) conjugates . SuccessA genome-wide RNA profiling and differential analysis (GWRPD) of sequence variations in 1557 different variants of SARS-CoV-2 was performed. This analysis resulted in the identification of five sites with 98–99% conservation among all analyzed variants, as indicated by the red segments in the outer ring of the GWRPD map A.Parallel bioinformatic predictions identified a priori potential G-C/A-T contents in the percentage bases for SARS-CoV-2  was the homopurine/palindrome site, which was previously unidentified . With a The predicted structures produced by RNAfold are shown in Coronaviruses are highly structured and a significant number of secondary and tertiary RNA structures have been identified, not only in the untranslated regions (UTRs), but throughout the coding regions of the genome ,17,18. AAlthough most remain uncharacterized, the highly conserved nature of these RNA structures among coronaviruses suggests that they may have functional relevance, perhaps through ensuring genomic stability or long-range RNA–RNA interactions or RNA–protein interactions important for viral replication ,17,18,26The region of interest identified here, referred to as the target site, is located in the 5′-end coding region of ORF1a/b and the nsp3 gene. ORF1a and ORF1b share the same 5′ start codon, but the FSE partially disrupts recognition of the stop codon, resulting in the replicase polyproteins, pp1a and pp1ab . ORF1a aThe specific variants, target site sequence, and the loci at which they begin are shown in Furthermore, the isolated target sequence within the previously mentioned variants of concern from Brazil, the United Kingdom, South Africa, and India were folded using RNAfold. The second variants from each region in In an effort to determine the relative importance of the stem-loop structure, specifically the partial homopurine palindrome site, in regulating the activity of SARS-CoV-2, this site was targeted by TFO. As mentioned previously, Kierzek et al. targeted a similar stem-loop in Influenza A virus with TFO, which resulted in an antiviral effect demonstrated by cell culture assays ,15. RegaThe target site can be characterized using techniques commonly used to determine RNA structure, including nuclease mapping, circular dichroism (CD) spectroscopy, and UV thermal melting of the isolated sequence. Nucleases capable of cleaving bases not involved in a double-stranded segment of the RNA sequence, such as those involved in the bulges and loop, can be used to digest the stem–loop structure. The produced segments can then be separated and identified through the use of gel electrophoresis. This will produce a unique band pattern in the gel which can be used to identify the presence of specific structures within the target site. The structures identified by the digest can then be compared with the original RNAfold-predicted structure. The band pattern obtained from this experiment will then serve as a control for future experiments involving the target site. The other proposed characterization methods to determine the structure of the isolated target site in vitro involve obtaining the UV melting curve and CD spectrum through the use of a SpectraMax i3x Multi-Mode Plate Reader and CD spectrometer, respectively. The shape of the produced melting curve can provide information on the stability of the structure, G-C content, reversibility of the melting process, and presence of the secondary structure within the sample . AdditioUpon characterization of the target site, the proposed triplex-forming region of the target site was targeted using a nanoparticle-delivered TFO. To determine whether the TFO was able to bind to the target site as predicted and to provide insight to optimal conditions which promote triplex formation, CD spectra, thermal melting points, and gel electrophoresis gel shift assay were performed and compared to data collected from target site characterization and any pre-existing literature. Each of these techniques are commonly used in RNA interaction studies.CD spectra and thermal melting points were obtained through the use of a CD spectrometer and SpectraMax i3x Multi-Mode Plate Reader, respectively. CD spectra of the target site and TFO as well as of the target site and TFO-nanoparticle conjugate were collected under the same conditions used to characterize the structure formed by the target site alone. Significant changes in either the location or intensity of the peaks of the CD spectra of the target site in the presence of TFO from that of the target site in the absence of TFO confirmed interaction between the two, a concept previously demonstrated by Kierzek et al. . From thf in the gel compared to the band from nuclease digestion of the target site in the absence of TFO.Gel electrophoresis can be used to determine optimal ratios which promote TFO binding, and thus, triplex formation. The target structure in the absence of TFO was used as a control to establish the standard band location within the gel. The band pattern of samples containing both the target structure and TFO at varying concentrations can be used to determine whether the TFO was able to interact with and bind to the target structure (indicated by a band shift closer to the wells) or not (no band shift from control). Additionally, the information provided by the gel can be used to determine the binding affinity of the TFO to the target site . NucleasAs previously mentioned, nucleic acids can be targeted using ASO and TFO methods. However, due to their instability, free nucleic acid therapeutics are not available in the absence of a nanoparticle stabilizer or carrier. Another method to target nucleic acids involves the use of aptamers. Aptamers can bind proteins and nucleic acid sequences with high selectivity and strong interactions. This technology can be applied in diagnostics, drug delivery systems, and therapeutics. Depending on the protein targeted by the aptamer, these molecules can have a wide range of applications. Aptamers can increase the selectivity of an antiviral particle or possess antiviral activity itself. The aptamer IBRV-A4 was able to prevent infection of bovine herpesvirus 1 in cell culture trials by preventing cellular uptake of the virus . HoweverLipid-based and polymer-based nanoparticles are often used as vehicles to deliver aptamers because they can transport large payloads of charged molecules  through the cell membrane using cell-penetrating peptides ,39,40. NTFOs can be conjugated to the surface or incorporated into the core of NPs and, in combination with other ligands, can improve the stability and targeting capabilities of the NP conjugate. Each of the previously discussed nanoparticle delivery methods is summarized in the context of TFO and stem-loop delivery in the graphical abstract at the beginning of this paper. As shown in the graphical abstract, delivering either the TFO or stem–loop structure to a cell infected with SARS-CoV-2 can potentially inhibit further viral activity, especially in the context of an antiviral nanoparticle such as MnZnS. Synthetic stem-loop structures can act as decoys for viral and host proteins, limiting those available to interact with the fully intact viral genome. Alternatively, TFOs can be delivered to virally infected cells, with the potential to form a triplex with the target site stem–loop in the SARS-CoV-2 genome and interfere with the genome’s natural folding in vivo.www.1DATA.life)—was last accessed 15 July 2021 –and then aligned in MATLAB , MAFFT (Berkeley Software Distribution), and MEGA (Pennsylvania State University) for multiple sequence alignment and Bayesian inference phylogenetics from a reinforcement learning approach using a progressive method and the Thompson–Higgins–Gibson (THG) method [Canis lupus familiaris (1), Felis catus (1), Mustela lutreola (7), and Panthera tigris jacksoni (1) [To achieve the data visualization shown in ) method ,44,45. Tsoni (1) . The strDue to its high conservation among variants, the structure of the partial homopurine palindrome site was explored using the RNAfold WebServer online program. The program is capable of folding up to 7500 nucleotides at a time. The target site was located between nucleotides 3179 and 3198; therefore, the genome was not folded past nucleotide 7765. Multiple folds were performed, using different lengths and regions of the SARS-CoV-2 genome, with the focus being placed on the region surrounding the target site.To further demonstrate its potential significance, the sequence of the partial homopurine palindrome target site was compared in SARS-CoV-2 variants of concern identified by the Centers for Disease Control and Prevention: the alpha variant (B.1.1.7), which originated in the United Kingdom; the beta variant (B.1.351), which originated in South Africa; the delta variant (B.1.617.2), which originated in India; and the gamma variant (P.1), which originated in Brazil. Each of these variants has been a source of increasing concern on a national and global scale as they have spread outside their country of origin, further exacerbating the effects of the pandemic. The Wuhan isolate served as the reference sequence for this comparison. Isolate sequences for each variant and the Wuhan reference isolate were obtained through the NCBI SARS-CoV-2 Nucleotide Records provided by the National Institutes of Health. The searches were performed periodically during the months of May 2020 through July 2020. Of the isolates included in this paper, collection dates ranged from December 2019 (GenBank accession number NC_045512.2) to June 2021 (GenBank accession number MZ558086.1).Although recent vaccines have proven effective in helping to counteract the SARS-CoV-2 pandemic, variants have emerged, resulting in concern regarding the ability of the virus to develop resistance to available vaccines and antiviral drugs in development. RNA-targeted nanomedicines are a potential solution to this problem. Recently, a partial homopurine palindrome site within the coding region of the SARS-CoV-2 genome has been identified. GWRPD analysis showed that this site has a greater than 99% conservation among more than 1500 variants, including those which have recently emerged and demonstrated high infectiousness in the regions of Brazil, the United Kingdom, South Africa, and India. This site was further explored using RNAfold to predict the potential structure formed by this region of the genome in vivo. The resulting stem–loop structure can be targeted with a TFO delivered by nanoparticles. Depending on the nanoparticle used, the TFO can be stabilized and protected from the body while being delivered in high concentrations to target cells. However, before this approach can be explored, the target site structure must be characterized in order to more accurately understand how it interacts with the nanoparticle-TFO delivery system. Additionally, although targeting this unique structure with TFO is an attractive method, nanoparticle-mediated delivery of ASO or aptamer targeting this or other highly conserved sites are excellent alternative strategies to consider.Disc. 2021-047; Our Docket No. 19484. mRNA SARS-CoV-2 vaccine construct. Inventors, Robert K. DeLong, Waithaka Mwangi and Juergen Richt. mRNA Vaccine Formulations and Methods of Using the Same. Kansas State University.


The COVID-19 pandemic has now strengthened its hold on human health and coronavirus’ lethal existence does not seem to be going away soon. In this regard, the optimization of reported information for understanding the mechanistic insights that facilitate the discovery towards new therapeutics is an unmet need. Remdesivir (RDV) is established to inhibit RNA-dependent RNA polymerase (RdRp) in distinct viral families including Ebola and SARS-CoV-2. Therefore, its derivatives have the potential to become a broad-spectrum antiviral agent effective against many other RNA viruses. In this study, we performed comparative analysis of RDV, RMP (RDV monophosphate), and RTP (RDV triphosphate) to undermine the inhibition mechanism caused by RTP as it is a metabolically active form of RDV. The MD results indicated that RTP rearranges itself from its initial RMP-pose at the catalytic site towards NTP entry site, however, RMP stays at the catalytic site. The thermodynamic profiling and free-energy analysis revealed that a stable pose of RTP at NTP entrance site seems critical to modulate the inhibition as its binding strength improved more than its initial RMP-pose obtained from docking at the catalytic site. We found that RTP not only occupies the residues K545, R553, and R555, essential to escorting NTP towards the catalytic site, but also interacts with other residues D618, P620, K621, R624, K798, and R836 that contribute significantly to its stability. From the interaction fingerprinting it is revealed that the RTP interact with basic and conserved residues that are detrimental for the RdRp activity, therefore it possibly perturbed the catalytic site and blocked the NTP entrance site considerably. Overall, we are highlighting the RTP binding pose and key residues that render the SARS-CoV-2 RdRp inactive, paving crucial insights towards the discovery of potent inhibitors. SARS-CoV-2 genome consists of a (+) ssRNA virus and its RNA-dependent RNA polymerase (RdRp) which help in viral replication and synthesis of its genome . RdRp ha759SDD761 , showing intermolecular interactions between RMP, primer-template, and RdRp . It was 59SDD761 . Motifs 59SDD761 . The resThe arrival of SARS-CoV-2 RdRp crystal structures of bound RMP (PDB ID: 7BV2) and APO (PDB ID: 7BV1) has provided an opportunity to elucidate their complex behavior and to understand the comprehensive inhibition mechanism. The recent studies are based on understanding the dynamicity of the modeled RdRp and its interaction with either RDV  or RTP . In the The protein structure of the SARS-CoV-2 RdRp was retrieved from the RCSB protein data bank . We also included crystal structures of HCV, BVDV, and DENV RdRp (PDB-ID:4NLD (for T1 and P2 site), 3FRZ (for T2 site), 4EAW (P1 site), 5I3Q , and 3FRZ, 5IQ6, and 1S48 (for Template entrance site)  to identTo understand the broader spectrum of the catalytic site as well as the NTP entry site, we included RDV as a parent molecule in our study. Since no bound crystal with RDV is yet reported, we performed targeted docking  of RDV a2+ ions. Model systems by Allner et al. and Carlson et al. were used for ions and triphosphate group, respectively  on each system is evaluated as follows:com, Grec, and Glig are the absolute free energies of complex, receptor, and ligand respectively, arranged over the equilibrium trajectory. The free energy, G, for each species can be calculated by using MM-GBSA and MM-PBSA approaches as follows:gas describes the gas phase energy and is the sum of internal energy (Eint), van der Waals interaction energy (Evdw), and electrostatic interaction energy (Eele). It is calculated using the parameters described in the AMBERff14SB force field  is the polar solvation contribution calculated by solving the Poisson-Boltzmann (PB) and Generalised-Boltzmann (GB) equations ) were obtained by sum of the Eele and )  and by determining the solvent-accessible surface area (SAS) using a water probe radius of 1.4 Å ) were calculated by Evdw and Gsol-np,PB(GB) ) . Gsol-npof 1.4 Å . The diep,PB(GB) . The binp,PB(GB) .−1) and (−5 kTe−1) was taken for positive and negative potentials, respectively, to generate the iso-surface of the protein.The calculations were done using the APBS program  in VMD  for finai and qj are the Cartesian coordinates of all Cα atoms and N is the number of Cα atoms used in building the matrix C. The < > sign indicates the ensemble average of the atomic positions in the Cartesian space. The resulting principal components (eigenvectors) were ranked by the corresponding total motion captured by them. In the end, there will be 3N eigenvectors generated, where N is the number of Cα atoms used in the building matrix. Porcupine graphs were generated using the “nmode” files obtained after PCA analysis. It basically provides the residue-based mobility plots for each eigenvector. Further, the free energy landscape was performed to explore the conformation change of proteins based on the PCA by using the ‘g_sham’ module  is the probability distribution of the conformation ensemble along the PCs.PCA was implemented to undermine the conformational changes in RdRp protein and especially essential motions were captured in APO and complex systems. The PCA analysis was performed to highlight the essential motions in the proteins . The PCA’ module . The frehot-spot amino acids in the RTP complex, we have performed computational alanine scanning for the residues highlighted in residue-wise energy decomposition results. The calculations were run on 100 frames from the last 100 ns of MD trajectory by using the MM-GBSA approach. In this method, an amino acid of interest is replaced with alanine and relative binding free energy is recalculated. Finally, the difference in the binding free energies of the wild type and mutant, ΔΔGbind, was computed as follows:To confirm the hot-spot residues were generated by using the maestro module.Negative values of ΔΔG bind indicate the favorable contributions of residues in wild type while positive values indicate the unfavourable contributions. The mutant models of all the site score, size, exposure score, enclosure, hydrophobic/hydrophilic character, contact, and donor/acceptor character. As per Halgren’s analysis, the average number of sites for sub-micromolar sites is 132, where lower exposure scores of 0.52 and higher exposure scores of 0.76 on average are considered better for sub-micromolar sites. For the donor/acceptor character and site score, the average for the sub-micromolar sites is 0.76 and 1.01, respectively. Druggability of the site is denoted by Dscore. Dscore values provide a rough estimate of whether the site is druggable. These scores were derived by To map the potential allosteric sites, the SiteMap  module iAll the images were generated using VMD  and grapMolecular docking has revealed the most likely binding pose of RDV and RTP. Targeted docking followed by focused docking has provided their final binding pose at the RMP bound site. The cluster representative of −7.5 kcal/mol and −8.9 kcal/mol docking energy were found for RDV and RTP, respectively . The supProtein structures must be converged in order to achieve robust results from MD simulations. The quantification of RMSD, RMSF, and Radius of gyrations are good indicators of the overall stability of any protein system. The APO-vs.-COM RMSD comparison reveals that the systems COM-RMP and COM-RDV are achieving stability after ∼20 ns of simulation while system COM-RTP was evolving and did not attain stability initially. The extension of COM-RTP simulation reveals that the system was stable in its next production stretch of 200 ns . However2+ ion and thus gained stability in the later phase of simulation  and one from motif E (C813)  and motif D (K798) which were found missing in its docked state (The stability of chosen ligands are thoroughly observed by understanding the interaction with residues at their most stable states obtained from their respective MD trajectories . By compE (C813) . SimilarE (C813) . RDV maiE (C813) . The RDVE (C813)  and its ed state  and in oAfter exploring the transitions, we further tried to understand the characteristics of the ligand’s respective binding pocket. So, RMP was chosen as the preferred active site and was surrounded with twelve residues: four hydrophobic residues , five polar  and three acidic residues  . At its The RMP and RTP form six hydrogen bonds, while RDV forms four hydrogen bonds. Residues that were crossing 30% cut-off in hydrogen bond occupancy were considered for this study. RMP forms hydrogen bonds with S682, G683, D684, A685, T687, and S759  and theiThe movements of all ligands can be justified after observing the hydrogen bond distance timeline of each residue . InitialMoreover, further thermodynamics analysis forms a strong base and might complement the binding pattern observations obtained from residue mapping and hydrogen bond analysis.bind = −26.37 kcal/mol, which is higher than RDV ∆Gbind = −23.83 kcal/mol and RMP ∆Gbind = −22.67 kcal/mol   . It can cal/mol)  and thiscal/mol) , RTP migAdditionally, we have characterized the pocket based on contributing residues and its respective chemical nature that might be crucial for designing ligands. Falling from RMP to RTP, it can be observed that RTP site (MD pose) is “basic rich” as residues K545, R553, R555, K621, R624, K798, and R836 are the significant contributors  while inElectrostatic potential was evaluated for all three systems in order to understand electrostatic complementarity between ligands and protein . ComplemThe final protein-ligand analysis justifies the RTP stability at NTP entry channel as it gains perfect complementarity from protein while the other two clearly miss this gain from their respective preferable site. This analysis also shows that RTP-MD pose is the best pose.To capture the significant conformational changes, PCA was applied to all the simulated systems. The first two PCs were found as important components for capturing the maximum variance in the Cα displacement during the MD simulations. This was found on inspection of the path of the trajectory connecting the minima’s and the sub-conformational spaces of different systems. The minima are numbered as per the appearance of the basins w.r.t. time during MD simulations. We found three minimas in APO systems, while only two minimas are observed in all complex systems . APO sysOverall, COM-RTP displays different behavior as compared to RMP/RDV systems. Two major regions have been identified through PCA and FEL analysis. Thumb subdomain is crucial for template entry while NiRAN domain is known for its interaction with other protein partners . COM-RTPThe key residues K545, R553, and R555 that are crucial for NTP entry channel have shown a remarkable drop in their contribution to RTP stability after they were mutated to alanine . ResidueT1 site: The general APO HCV RdRp consists of a Δ1 loop tucked on the thumb domain which is displaced by non-nucleoside inhibitors (NNIs) and forms a hydrophobic T1 pocket  (bdomain) , we founbdomain) . TherefoP2 site: The residues V588, L602, M756, I757, D761, A762, V763, C813, and S814 of CoV-2 RdRp are found to be aligned with the residues G192, L204, L314, V315, D319, L320, V321, C366, and S367 of HCV RdRp  , we founral RdRp .TES site: Since the RNA template entrance site (TES) exists in all polymerases and has been well explored in DENV, BVDV, and HCV RdRps, this site could also be targeted for designing NNIs. We have aligned all these four RdRps and highlighted the conserved residues accordingly. We found more number of conserved residues in BVDV RdRp than others with CoV-2 RdRp. In CoV-2, the conserved residues are N497, A502, G503, F504, K545, R553, A554, R555, V557, S682, G683, A685, and T687  and its metabolites, RMP , and RTP (active form) was performed to understand the mechanism of RdRp inhibition, the reason behind the active form of RTP only, and its interaction pattern with key residues. Previous studies with RTP or RDV were performed on homology models and after the arrival of crystal structure of SARS-CoV-2 RdRp, we implemented a holistic approach to extract the meaningful insights that lead to inhibition. RMP was bound in crystal, however as stated by Yin et al. in 2020, the metabolite RTP is known to be the active form of RDV . TherefoTP entry . This ou


The scaffolding protein gephyrin is a core component of many inhibitory synapses. Bai and colleagues now show that phase separation of gephyrin with the subunits of both glycine and GABA receptors underlies the formation of postsynaptic sheets at the inhibitory synapses.AR).1To assure precise processing of information, contacts between neurons require the alignment of the release sites, presynaptic terminals, with the ionotropic transmitter receptors at the postsynaptic plasma membrane. Downstream of these receptors, scaffold molecules, kinases and cytoskeleton components all orchestrate the signaling cascade induced by the neurotransmitter binding and ion influx. Each neuron in the mammalian central nervous system relies on both the excitatory and inhibitory inputs. A long-standing view is that the postsynaptic density (PSD) represents a hallmark of the excitatory synapses. Recent cryo-electron tomography work suggested that even the inhibitory synapses contain thin-sheet densities of ~5 nm, so-called iPSD, underneath the clusters of glycine receptor (GlyR) and type-A γ-aminobutyric acid receptor polymers separate from homogenous aqueous mixtures forming fluid condensates.4 In the study published in Cell Research, Bai and colleagues examine whether the principles of LLPS might underlie the formation of iPSDs,5 focusing on the interaction between transmembrane receptors and gephyrin, a well-known scaffold protein of inhibitory synapses.It is emerging that the principles of liquid-liquid phase separation (LLPS) could explain the mechanisms behind the formation and regulation of the PSD at the excitatory synapses.AR6 and contains an N-terminal trimerization G-domain (GPHN-G) and a C-terminal dimerization E-domain (GPHN-E) linked by a flexible C-domain. GlyR and GABAAR, via their intracellular loops, bind to the E-domain of gephyrin.7 Traditionally, gephyrin is considered to form hexagonal lattice crosslinking the receptors at the plasma membrane and connecting them with the cytoskeleton and downstream signaling molecules.7 Here, the authors show that the loop of GlyR β subunit fused to GCN4 coiled-coil (GlyR-β) and E-domain of gephyrin (GPHN-E) form liquid condensates in solution (at low µM concentrations) and at the supported membrane (in the nM range).5 The ability of GlyR-β to oligomerize plays an important role in its capability to phase separate with GPHN-E. Besides GlyR-β, TM3–4 loops of GABAAR α3-subunit also phase separates with GPHN-E suggesting liquid condensation to be a general feature of inhibitory synapses  and as a bait to recruit partner proteins for specific downstream biological function. Finally, this study breaks with the dogma that PSDs are only a feature of excitatory synapses. The authors reconstitute the proteinaceous sheets observed at the inhibitory synapses and show them to be formed by a similar mechanism as disc-shaped structures at the excitatory postsynapse.There are several implications of this study. First, the observation that different regions of the same proteins (GlyR-β and GPHN-E) are important for phase separation or direct binding, suggests that these two processes could be regulated separately. The positively charged region of GlyR-β (a.a. 354–383) and negatively charged surface at the subdomain II of GPHN-E are essential for condensate formation, despite hydrophobic interactions being critical for their direct binding. Indeed, the authors show that increasing salt concentration does not disrupt the binding between GlyR-β and GPHN-E, but it prevents phase separation. Moreover, the full-length gephyrin has a reduced ability to form liquid condensates. The authors mapped two inhibitory peptide sequences (a.a. 259–274 and a.a. 303–318) within the linker region of the C-domain. Interestingly, the dynein light chain, which is known to interact precisely with the C-domain of gephyrin,AR subunits can cluster independently of gephyrin,10 implying that other factors and scaffold proteins, e.g., collybistin,11 might be the additional mediators of the postsynaptic phase formation at the inhibitory synapses. Finally, in pathology, some patients with autism spectrum disorder, schizophrenia and epilepsy contain mutations in GPHN gene that encodes the G-domain of gephyrin.12 It remains to be determined to what extent these mutations and deletions of gephyrin alter its phase separation properties and potentially modulate the molecular basis of these complex diseases.As it often happens, this study opens a range of interesting questions. What is the effect of phosphorylation on the gephyrin-mediated phase? The authors generated phosphomutants in different regions of gephyrin’s C-domain, showing that phosphorylation of various sites can either enhance or reduce the condensate formation. Further studies will be needed to identify how specific kinases regulate these different phosphorylation sites during neuronal activity. Another critical point is that GABA


Screws are the most frequently inserted orthopaedic implants. Biomechanical, laboratory-based studies are used to provide a controlled environment to investigate revolutionary and evolutionary improvements in orthopaedic techniques. Predominantly, biomechanical trained, non-surgically practicing researchers perform these studies, whilst it will be orthopaedic surgeons who will put these procedures into practice on patients. Limited data exist on the comparative performance of surgically and non-surgically trained biomechanical researchers when inserting screws. Furthermore, any variation in performance by surgeons and/or biomechanical researchers may create an underappreciated confounder to biomechanical research findings. This study aimed to identify the differences between surgically and non-surgically trained biomechanical researchers’ achieved screw tightness and stripping rates with different fixation methods.Ten orthopaedic surgeons and 10 researchers inserted 60 cortical screws each into artificial bone, for three different screw diameters , with 50% of screws inserted through plates and 50% through washers. Screw tightness, screw hole stripping rates and confidence in screw purchase were recorded. Three members of each group also inserted 30 screws using an augmented screwdriver, which indicated when optimum tightness was achieved.n = 928, 95% CI 81–83) and 76%  respectively (p < 0.001); surgeons stripped 48% (872/1800) of inserted screws and researchers 18% (330/1800). Using washers was associated with increased tightness  compared to screws inserted through plates  (p < 0.001). Researchers were more accurate in their overall assessment of good screw insertion (86% vs. 62%). No learning effect occurred when comparing screw tightness for the first 10 insertions against the last 10 insertions for any condition (p = 0.058–0.821). Augmented screwdrivers, indicating optimum tightness, reduced stripping rates from 34 to 21% (p < 0.001). Experience was not associated with improved performance in screw tightness or stripping rates for either group (p = 0.385–0.965).Unstripped screw tightness for orthopaedic surgeons and researchers was 82% (Surgeons and researchers showed different screw tightness under the same in vitro conditions, with greater rates of screw hole stripping by surgeons. This may have important implications for the reproducibility and transferability of research findings from different settings depending on who undertakes the experiments. Screws are the most commonly used orthopaedic implant and are needed in the majority of orthopaedic operations. In current practice, non-locking screws require the user’s subjective assessment of the torque that should be applied to achieve optimum fixation. Analysis of surgical techniques has shown a concerning spectrum of abilities in creating adequate constructs for osteosynthesis . Screw iThis study was designed to assess the following comparisons between a sample of orthopaedic surgeons and biomechanical researchers, with the null hypothesis of there being no difference in their performance under any defined conditions. Comparisons were made to investigate any differences in the groups’ screw tightness and screw hole stripping rates when inserting screws into plates or through washers and when inserting different diameter screws. Additional comparisons were made to ascertain any difference between surgeons and researchers in: the reported confidence in screw insertions that had or had not stripped screw holes, the detection of stripping of screw holes, the presence of a learning effect when inserting screws, the impact of awareness of applied torque and whether indication of optimum tightness affected screw tightness and stripping rates.Custom made testing apparatus was created for standardised screw insertion. Artificial bone sheets  were manufactured, 4 mm thick, with a density of 20 pounds per cubic foot (PCF). Using a milling machine , 90 drill holes were made perpendicularly in each of 40 sheets; each sheet contained 30 drill holes of 2.0 mm, 2.5 mm and 3.2 mm to receive 2.7 mm, 3.5 mm and 4.5 mm cortical screws, respectively. A wooden jig was created, containing a foam base to mimic the stiffness of human soft tissue  set to vibrate and alarm when the optimum tightness was reached; optimum tightness was set at 70% of the mean average stripping torque , 10, whit tests for comparisons of years of experience, screw tightness and stripping rates for surgeons and researchers, and paired two-tailed t tests for comparisons between tested variables: for plates and washers, for different screw diameters, for reported confidence for stripped and unstripped insertions, for the first ten screw insertions against the last ten screw insertions and for unaugmented and augmented screw insertions. Rates of screw hole stripping were compared using McNemar and Chi Squared tests. Bonferroni corrections were performed for cases of multiple comparisons, with adjusted values reported. Using the confidence values reported, the sensitivity, specificity and accuracy for screw hole stripping were calculated. The maximum and mean average Youden Indices were calculated. Results were considered significant at a level of significance of 0.05, and confidence intervals were calculated at 95%. Statistical tests were performed with IBM SPSS Statistics for Windows, version 20 . All data are available in an online repository  than for researchers  (p < 0.001), with a greater stripping rate: 48% (872/1800) versus 18% (330/1800) (p < 0.001). Tightness and stripping rates for different screw diameters and plate and washer insertions are summarised in Table p < 0.001). Lower tightness was seen with 4.5 mm insertions compared to 3.5 mm insertions for both surgeons (p < 0.001) and researchers (p = 0.04) and compared to 2.7 mm insertions for researchers (p < 0.001). Analysed separately, for surgeons and researchers, there was no association between experience and either screw tightness  or stripping rates  , –83, n = p < 0.001), researchers—7.4 versus 5.1 (p < 0.001) . Both groups were similarly specific when correctly predicting an unstripped screw hole—95% for researchers and 91% for surgeons    and researchers (p = 0.001) (Table There was no significant change in screw tightness between the first 10 and last 10 screws inserted for any screw diameter or fixation technique 21) Fig. . A stron90) Fig. . Using a1 Fig. . Within this study, surgeons showed a different ability from researchers in controlling screw insertion. There was a spectrum of abilities within both groups, with some surgeons and researchers generating very consistent screw tightness and minimal stripping rates, though both groups had participants who were insensitive to detecting stripping. Our findings raise concerns about the validity of methods in biomechanical research when insertion torque is neither recorded nor reported given the rates of poor screw insertion. Studies exclusively involving surgeons may generate more clinically transferable findings by mimicking clinical conditions more accurately; however the higher rate of stripped insertions that might occur during the experimentation could introduce into the methods an underappreciated confounder given the reduced compression generated and reduced pullout strength of stripped screws , 10 and This is the first study comparing tightness and stripping rates for different fixation methods and screw diameters. The same stripping rate was seen for both plate and washer fixation with average unstripped tightness close to the optimum tightness, defined as being between 70 and 80% of the stripping torque , 10. SmaGood screw fixation is reliant on the ability to contemporaneously critique a screw’s insertion to ensure the screw will perform as intended. If insertion is felt to be poor, alternative remedies, though often suboptimal, can be enacted if the screw hole has been stripped. These corrections include, for example, re-siting a screw or inserting a larger diameter screw. Both groups in this study, on average, correctly showed a significant difference in the confidence of a screw’s holding ability between unstripped and stripped screw insertions. However, researchers were appropriately less confident when screw holes were stripped. Building on the need for sensitivity to inappropriate tightening, the capability to determine when a screw insertion was stripped differed between researchers and surgeons. Accuracy in detecting stripping highlighted that some participants were insensitive to stripping, a finding seen before by Stoesz et al., who found that more than 90% of stripped screw insertions went undetected by surgeons . AdditioThere was weak evidence of increasing tightness with more insertions, with no change in the stripping rate between the comparative groups of the first third and last third of insertions. This echoes the findings of a larger study into screw insertion variables that showed for all but a few of the tested conditions there was no increase in tightness with more insertions, and that the performance when inserting the first 10 screws was representative of a larger number of screw insertions . More sc3 artificial bone models finding a significant (p < 0.001) reduction in the stripping rate from 42 to 15% when they were unblinded to the applied torque [Awareness of the applied torque value and when optimum torque has been reached was seen to reduce stripping rates. Gustafson et al. investigated surgeons inserting screws into 0.1 g/cmd torque . Bone chd torque , 10. Usin = 3960). The similarity between the tightness of the first ten screws inserted and the last 10 screws for a test variable shows that future studies may appropriately investigate a situation with the insertion of only ten screws. However, even ten screws under the same conditions are more than the number used in most previous biomechanical studies into screw fixation [One of the key strengths of this study is the number of screws inserted, and thus the power of this study, as this is considerably more than any previous work examining screw insertion outcomes . The traThere were limitations with the study, including that the model used for testing mimicked low density bone, with only unicortical fixation performed which may not be representative of the majority of screw insertions in clinical practice. However, previous work has shown that screw techniques in human cadaveric models mimic those of artificial bone . UnicortThe sample of surgeons and researchers analysed frequently showed different screw tightness under the same conditions, with significantly greater rates of screw hole stripping by surgeons. With the majority of screw research being performed by non-surgical, biomechanical researchers, there may be a failure to replicate in vitro findings if the skills of the surgeons differ greater from those making research discoveries. Greater attention to teaching optimal screw insertion to both surgeons and researchers is warranted alongside further investigation into the clinical use of augmented screwdrivers to indicate optimum tightness.


Such data will be useful inthe interpretation of observational data from the James Webb SpaceTelescope. Using an ultrahigh vacuum apparatus, we conducted reflection–absorptioninfrared spectroscopy measurements in the mid-IR range of pure methaneice and methane-containing ice mixtures of interest to interstellarand solar system ice chemistry, e.g., with H2O and N2 molecules. We found that nuclear spin conversion (NSC) insolid methane and its crystalline structures is affected—indifferent ways—by the presence of H2O and N2. Specifically, we found a relationship between the thicknessand the solid-state ordering transformation in methane thin films.This new study of the NSC of pure CH4 ice and of the CH4:H2O ice mixture at 7 K is carried out in relationto the segregation of H2O using the ν1 and ν2 IR inactive modes of methane. The diffusionof N2 and CH4 in the CH4:N2 ice mixture with temperature cycling has been studied to obtainthe relationship between IR features and the state of aggregationof the ice.Mid-infrared studiesof fundamental modes of ices of pure CH Methane is found at the single digit percentlevel in interstellar ices4 but is abundantin outer solar system bodies, such as Titan, Triton, Pluto, and others.Its importance is related to the fact that is considered a key moleculein the development of prebiotic life.9Solid methane and its mixtures with othervolatiles are presentin ocean beds as clathrate hydrates,10 PhaseI is the equilibrium phase at T > 20.4 K. It isaface-centered cubic (fcc) crystal with orientationally disorderedCH4 molecules. Phase II is stable below 20.4 K and consistsof an fcc lattice with eight ferrorientational sublattices . Thus, in the primitive cell, the six CH4 thatare orientationally ordered are subject to both the crystalline andorientational fields, while at the location of the two virtually freerotators, the octupolar orientational field vanishes.11 Recently, a metastable phase of solid methane at T < 7.0 K was discovered; it is a crystalline phase withorientational order between Phase II and Phase I.12At cryogenictemperatures and low pressures, there are two recognizedand well-documented solid phases.–1  and a deformation mode (ν4) at 1302 cm–1 (7.68 μm). In addition, methane solid goesthrough nuclear spin conversion (NSC) at cryogenic temperatures15 where the change in nuclear spin configuration influences the ro-vibrationalspectrum. Molecular-level interaction between CH4 and H2O is accompanied by significant shifts in peak positions andspectral features in the near16 and mid-IR17 spectral regions. Specifically, the interactionof water with methane manifests itself in the detection of two additionalmodes, ν1 and ν2, which are inactivein pure solid methane.19In the mid-IR, methane has two active vibrationalmodes, a C–Hstretching at 3009 cm21 at low concentrationsin water-dominated ices; when ejected in thegas phase it leads to a warm carbon-chain chemistry22 and the production of unsaturated hydrocarbons.23 In planetary systems, it is found in outer solarsystem bodies,24 in satellites of planets,25 and in water-rich comets.26 Near-IR features of methane ice on outer solar system bodiesare blue-shifted,27 indicating that CH4 is mixed with N2, the other major component inthose ices. The absence of combination modes  involving IR inactive modes ν1 and ν2 in Triton’s spectrum has been used to infer that CH4 molecules are isolated and dispersed in N2:CH4 ice.30 A study of the shift positions and broadening ofmid-IR spectral features of CH4 in CH4:H2O mixtures grown at 30 K is given in Gálvez et al.(2009).31 CH4:H2Omixtures in the 14–60 K range, and in particular the inactiveν1, are studied in Herrero et al. (2010).32 The possible origin of the activation of theν1 mode of CH4 in CH4:H2O mixtures is investigated by means of solid-state calculationsin Escribano et al. (2014).33Methane has been detectedin the interstellar medium (ISM)2O and N2. We aim to quantify how the presence of these molecules inmethane-rich ice produces changes in IR spectra. Furthermore, we wantto see whether the state of aggregation can be discerned from IR features,as it was done for CO–CO2 ice mixtures34 and CO2 films on water ice.35 We proceeded as follows. We first measured theeffect of deposition methods, thermal cycling, and deposition temperatureon thin films of pure methane in ultrahigh vacuum (UHV) conditions.We then measured the spectral changes that methane ice experiencesin the presence of H2O and N2. This allowedus to study the diffusion and segregation process in CH4:H2O and CH4:N2 ice mixtures.Thegoal of this work is to study the IR absorption features ofpure methane ice and methane ice mixture with H–10 Torr after a bake-out; see He et al.36 for details; here only the main features that are relevant to thisstudy are summarized. Gases were deposited on a gold-coated copperdisk mounted on a closed cycle cryocooler (ARS DE-204 4K). The temperatureof the sample was monitored and manipulated by a calibrated silicondiode placed behind the sample and paired with a Lakeshore 336 temperaturecontroller. A cartridge heater installed behind the sample can beused to heat the sample. By controlling the heating output of thecartridge heater, the temperature can be adjusted between 5 and 300K with an accuracy of better than 50 mK.Experiments were performed at SyracuseUniversity in an UHV chamberin which the pressure can routinely reach 4.5 × 1015 molecules/cm2) of ice were formed at a relatively high deposition rate of 100ML/min. This deposition dose was calculated using the impingementrate; see the Appendix of He et al.36 fordetails. For the deposition of CH4 and H2O,two separate UHV variable leak valves were used. Distilled water underwentat least three freeze–pump–thaw cycles before beingsent into the chamber. Premixed CH4 and N2 gaswas deposited using a single leak valve for CH4:N2 mixture deposition except for one occasion: a molecular beamlinewas used to deposit 100 ML of CH4 for the experiment presentedin Methane ices were grownon the sample disk by introducing methanethrough a stepper motor-controlled UHV variable leak valve. A LabVIEWprogram controlled the deposition rate and thickness. In all experiments,100 monolayers  configuration with an incident angleof ∼78° was used to obtain mid-IR spectra of the ice.Spectra were measured and averaged every 20 s at a resolution of 1cmWe carried out two sets of experiments. In the first set, we studiedthe IR signatures of crystalline phases of methane under differentconditions. For these experiments, pure methane ice of different thicknesseswas deposited at either 6 or 10 K. Then methane ice was either keptat a specific temperature for an extended period of time or heatedslowly at 3 K/min until the methane ice desorbed past 40 K. In thesecond set, methane ice mixed with either water or nitrogen was depositedat 10 K, and then the ice went through temperature cycling (heatingand cooling) to study the state of the aggregation of the ice throughchanges in IR features during the process.11 In such experiments, the transition happensabruptly at 20.4 K. However, from an analysis of methane thin filmsdeposited in the 7–30 K range, we observe that the transitionfrom Phase II to Phase I occurs over a temperature range. 4 and ν3 modes of pure methane ice depositedat different temperatures. At 7 K the ice is in Phase II*, which isa metastable phase without the presence of band splitting due to NSC.12 In pure methane ice, satellite peaks emergenear ν4 and ν3 due to IR-allowedtransitions brought about by conversion of the nuclear spins of thehydrogen atoms in CH4 ice.14 For methane deposited at 10 K, the ice is in crystalline Phase IIwith the fully emerged band splittings due to NSC,14 in agreement with the results presented in a previous publication.12 As we raise the deposition temperature, we observethat the band splitting gradually diminishes due to the disruptionof neighboring lattice sites. Between 20 and 22 K multiple peaks ofν4 and ν3 modes converge into asingle broad peak for each mode due to orientational disordering atlattice sites. There is a significant blue shift from 3010.6 to 3014.2cm–1 for the ν3 mode in that temperaturerange. Above 22 K, the ice forms an orientationally disordered fcclattice.Experiments to characterizethe Phase I–Phase II transitionin methane ice are typically conducted in closed cells.4 on a substrate at 10 K, and then heated thesample from 10 to 45 K at a rate of 0.1 K/s. The bending mode ν3 absorption spectra for all these thicknesses, normalizedto the maximum absorption of all the spectra for all the spectra ofthe same thickness during the heating process, are shown in 14 . We obtained the temperature range forthe phase transition based on an analysis of the shifting of the peakposition. The right panel of 3 peak,and the position is obtained for each spectrum during the warm-up.More details of the fitting scheme are described in Emtiaz et al.12 The peak position is then plotted as a functionof temperature and is shown in the right panel of –1/K as the starting point of the phase transition. The end point ofthe phase transition is more obvious from the plot. The temperaturerange for the phase transition for methane ices of different thicknessesis then shown in the left panel of 4 ice. These experimentssuggest that methane ice experiences thin-film effects for coveragebelow 15 ML. For higher coverage (≥100 ML), the ice is independentof thin-film effects.We deposited 5, 10, 15, 20, 30, and100 ML of CH12 As we increase thetemperature above 7 K, we observe an exponential increase in the relaxationrate up to 8.5 K.12 Depending on the depositionrate, we found different rates of NSC for methane ice. The left panelof 3 mode, which is a signof NSC in IR spectroscopy. On the other hand, the right panel of –1) for the ν3 mode and the Q(1) band (1297.8 cm–1) for the ν4 mode. As we keep the ice at 6 K for 12 h, we see that theband splitting is more pronounced. In the ice prepared slowly, orientationalordering at lattice sites takes place; therefore, we see a significantrate of NSC even at 6 K. This fact further strengthens our argumentthat at 6 K we observe a metastable phase of methane which is a crystallinephase with an orientational ordering between Phase I and Phase II.12We know that at 6 K the methane thin film is in a metastable phase;at that temperature nuclear spin conversion does not take place overlaboratory times.4 andν3 modes of pure methane and methane–watermixture with different mixing ratios. We observe a significant changein IR features and fwhm values for increasing amounts of water mixedwith methane. For CH4:H2O (=95:5) we observethat the peak position is red-shifted by 0.8 and 0.7 cm–1 for ν4 and ν3, respectively. Thefwhm becomes 14 and 7 cm–1 for the ν4 and ν3 modes, respectively, which is a significantincrease from pure methane. The red shift in the peak position ismore significant for the ν3 than for the ν4 mode. All the relevant values are listed in 4:H2O ice using the CH4 IR inactive modes. Pure methanehas two IR inactive modes: ν1 (2904.5 cm–1) and ν2 (1540.0 cm–1). When wateris mixed with methane, there is no band splitting due to NSC in PhaseII CH4:H2O mixtures at 10 K. However, the presenceof water activates the IR inactive modes.194:H2Oat two different stages of the same experiment. The dotted markedspectra represent CH4:H2O after the depositionat 10 K. The solid line shows the band after the ice is taken to 30K and then cooled back to 10 K. Two intense bands are observed at3500 cm–1 (not shown) and at 1635 cm–1, caused by O–H stretching and O–H–O scissors-bending,37 respectively. We observe significant changesin ν4 and ν3 modes before and aftertemperature cycling. Band splitting such as R(0) for ν3 and Q(1) for ν4 emerges after temperature cycling.Band strengths of ν1 and of O–H–O scissors-bendingdecrease by about 50% after the heating and cooling process. Thischange in IR features indicates that fewer CH4 moleculesare in contact with water molecules; therefore, a partial segregationof water in the CH4:H2O ice has taken place.This segregation process happens during orientational reordering oflattice field sites of methane molecules during temperature cycling.In this section we investigate the CH12 There is no NSC for CH4:H2O ice deposited at 10 K and kept at that temperature for 30 min.However, if the CH4:H2O ice is taken to 30 Kand then rapidly cooled to 7 K and kept for 100 min, NSC splittingis observed, as in the case of pure methane ice. The fact that theCH4:H2O ice mixture—when annealed at30 K—goes through the NSC process similar to pure methane ice(while the mixture quenched to 7 K does not) indicates that watersegregated and left patches of pure methane where NSC can take place.A similar segregation process has been observed in CO:CO2 = 9:1 ice mixtures when CO undergoes a phase transition from amorphousto crystalline.34 During the transition,CO2 molecules form clusters. The segregation of H2O from the CH4:H2O mixture during NSC may bea process that is common on icy bodies in the solar system. All therelevant values of ν3 and ν4 vibrationalmodes peak position and band shift are listed in Another piece of evidence of segregation is asfollows. Nuclearspin conversion occurs in methane ice between 7 and 11 K.2 is a nonpolar molecule, and the unitcell of N2 has a lattice parameter of 5.64 Å,38 which is close to the value of the lattice parameterof fcc methane solid, 5.89 Å. Thus, it is a reasonable assumptionthat in the CH4:N2 mixture, N2 isin a substitutional site rather than in an interstitial site as theices have similar lattice parameters and both have an fcc structure. 4 ν4 andν3 modes of CH4:N2 with differentmixing ratios. We observe that there is little change once we introducea small amount of N2 in the ice matrix. The CH4 lattice symmetry is disrupted once the impurity level reaches 20%.At the CH4:N2 = 80:20 ratio the satellite peaksof CH4 ν4 and ν3 modescompletely disappear which indicates that the N2 moleculesin the ice matrix cause enough disruption of the symmetry of the originalCH4 ice that the collective behavior giving rise to NSCis suppressed. We see a significant change in IR features and FWHMvalues in CH4:N2 = 80:20. In this case, thepeak position is red-shifted by 0.4 and 2.3 cm–1 for ν4 and ν3, respectively. Thefwhm becomes 9.4 cm–1 for ν4 and9.5 cm–1 for ν3 modes, which isa big increase from pure methane ν4 and ν3 modes, 4.1 and 6.3 cm–1, respectively.The blue shift in the peak position is more significant in the ν3 mode than in the ν4 mode. All the relevantvalues are listed in N4:N2 (=80:20)before and after thermal cycling from 10 to 20 K and back to 10 K.It suggests that there is a structural rearrangement in the ice mixtureas the ice mixture undergoes a heat cycling between 10 and 20 K. Weobserve a significant change in the ν3 mode and ν2 + ν4 and ν3 + ν4 combinations modes. The peak position of the ν3 mode blue-shifts 3012.5 cm–1 to 3016.3cm–1 , and the fwhm becomes 14.8 cm–1 from 9.5 cm–1. In the case of combination modes,ν2 + ν4 loses its band strengthby 50% and ν3 + ν4 blue-shifts.The broadening of the bands after the thermal cycling suggests thatthere has been a loss of symmetry or ordering. A detailed study ofthe CH4/N2 diffusion process will be presentedin a forthcoming work.4 in the solidstate in interstellar environments, the formation of CH4 aggregates likely requires thermal input for the diffusion of CH4 molecules, and we showed that IR features can be used tocharacterize the thermal history of the mixture. In planetary environments,including comets, the interaction of methane with water is important,as well the detection of IR features that are affected by thermalcycling. Our results show how thermal cycling of a CH4:H2O ice mixture induces the segregation of water and methaneand how this change can be followed by the analysis of IR features.As mentionedin the 39The orientational ordering transition(Phase II to PhaseI) in methane ice films occurs over a range of temperature in contrastwith measurements of bulk methane ice in closed cell experiments wherethe transition is abrupt.Nuclear spin conversion at cryogenic temperatures dependson the deposition rate. NSC is virtually absent in a fast depositionfrom background gas (100 ML/min), but it shows up in a slow depositionof ice at 6 K (1 ML/min) from the beamline.%) amount of water presentin the ice matrix causes changes in CH4 IR bands. The peakposition of the CH4:H2O matrix is red-shiftedas we increase the amount of water in the mixture. Similarly, thefwhm of the CH4:H2O broadens with the increasedpercentage of water.A small (54 bands (ν1) appear. Whenthe ice is taken to 30 K and then cooled back to 10 K, these forbiddenbands get greatly reduced (50% for a CH4:H4O= 80:20 mixture), indicating that there are fewer CH4 moleculesin contact with H2O molecules. This is an indication thatwater is partially segregated from CH4.The introductionof an amount of water greater than5% during deposition at low temperatures (10 K) suppresses NSC, andforbidden CH4:H2O mixtures,in CH4:N2 mixtures a much larger amount (20%)of N2 is required to suppress NSC. Once the N2 fraction is over 20%, there is a blue shift of peak positions ofν3 and ν4 modes. As the temperatureis raised, the orientational symmetry of the CH4 ice structurechanges.Compared to CHThe main findings of our measurements of mid-IRbands of pure methaneice and in mixtures with water and nitrogen are as follows:4 ice in interstellar and planetaryenvironments, such as its thermal history and its degree of mixingwith water and nitrogen molecules.Taken all together, these observationsshould help the interpretationof observations of CH


This study describes a new and safe freehand cervical pedicle screw insertion technique using preoperative computed tomography (CT) morphometric measurements as a guide and a medial pedicle pivot point (MPPP) during the procedure. This study included 271 pedicles at 216 cervical spine levels (mean: 4.75 pedicles per patient). A pedicle diameter (PD) ≥ 3.5 mm was the cut-off for pedicle screw fixation. The presence and grade of perforation were detected using postoperative CT scans, where perforations were graded as follows: 0, no perforation; 1, perforation < 0.875 mm; 2, perforation 0.875–1.75 mm; and 3, perforation > 1.75 mm. The surgical technique involved the use of an MPPP, which was the point at which the lines representing the depth of the lateral mass and total length of the pedicle intersected, deep in the lateral mass. The overall success rate was 96.3% . In total, 54 perforations occurred, among which 44 (81.5%) were Grade 1 and 10 (18.5%) were Grade 2. The most common perforation direction was medial . The freehand technique for cervical pedicle screw fixation using the MPPP may allow for a safe and accurate procedure in patients with a PD ≥3.5 mm. Cervical spine surgery is being performed with increasing frequency . In patiCurrently, with technological advances such as computer assisted navigation (CAN) systems such as Stealth Station Spine Surgery Imaging and Surgical Navigation with O-arm  or Ziehm Vision FD Vario 3-D with NaviPort integration , it has become possible to contribute to accurate posterior fixation ,12. HoweWe therefore developed a new freehand technique for cervical pedicle screw insertion; guided by preoperative computed tomography (CT), our procedure may provide accurate and safe screw insertion, with less exposure to intraoperative radiation. In this study, we aimed to describe this novel freehand cervical pedicle screw insertion technique—using a medial pedicle pivot point (MPPP)—through technical statements. We also examined the accuracy and safety of the technique regarding the presence and grade of pedicle screw perforations.Patient populationThis study was approved by our institutional review board and ethics committee. A standardized protocol was used for all enrolled patients, which included pre- and postoperative CT scans to measure anatomic parameters and the accuracy of pedicle screw placement. All procedures were performed in accordance with the relevant guidelines, regulations of our institute and a waiver regarding the need for informed consent. This prospective study included 57 patients  who underwent surgery with pedicle screws inserted via our novel freehand insertion technique between February 2018 and October 2020; all surgeries were performed by a single orthopedic spine surgeon. A pedicle diameter (PD) of 3.5 mm was the minimum cut-off value for 3.5 mm diameter pedicle screw fixation; if the PD was <3.5 mm, lateral mass screw fixation was utilized instead. In the case of abnormalities—such as a high-rising vertebral artery on preoperative CT—an alternative method was used, such as a lateral mass screw. Additionally, for cases in which a CT scan of an individual pedicle demonstrated sclerotic bony change with superior/inferior articular processes and a lack of cancellous track, lateral mass screws were used for conversion. Therefore, the number of pedicle screws did not always correspond with the number of surgically treated levels.Preoperative radiographic measurementsEach patient underwent preoperative plain cervical CT using a multisided scanner . Both the sagittal and axial views of cross-sectional images (1 mm cuts) were obtained in parallel with the pedicle trajectory. For quality control and CT standardization, technicians in the radiology department received regular instruction by the surgeon performing the operations regarding how to obtain correctly oriented sagittal and matched axial scans of the pedicles.To identify the correct starting point and direction of screw insertion for each pedicle, linear and angular parameters were measured on both the sagittal and axial CT images; this was performed for each patient at every cervical level on both the right and left sidesOn sagittal images, the distance (in mm) was measured from the tip of the inferior articular process of the cephalad cervical vertebra to a point in the lateral mass transected by a line drawn through the center of the pedicle; this was defined as the facet-pedicle distance FPD; A. The saIn the axial view, the starting point and designated MPPP were set first B. The laThe linear and angular radiologic parameters measured via the preoperative CT scan were captured at all cervical levels scheduled for surgical treatment; this was presented on a PowerPoint slide in the form of a screenshot, allowing the images to be viewed on a computer monitor in the operating room.Surgical techniqueAfter inducing general anesthesia, patients were placed in the prone position on a Jackson table , with their head placed on a Mayfield headrest  in a neutral position. Shoulders were pulled caudally and maintained on the sides using heavy bandages. The mainstay procedure for realignment of the cervical spine using a Gardner Wells tong was not performed in this study, as results may differ from the preoperative CT measurement. A lateral view radiograph was obtained to ensure that all necessary cervical levels could be visualized, and cervical lordosis was comparable to that preoperatively noted via CT. If the cervical lordosis adjusted with the Mayfield headrest was different from the lordosis observed on the preoperative CT scan, the headrest device was readjusted to match the preoperative image as much as possible.A posterior midline incision was made, and the posterior elements of the cervical levels undergoing instrumentation were subperiosteally exposed. The dissection was then extended laterally to expose the lateral borders of the lateral masses. PowerPoint slides containing measurements of the intended levels and pedicles, based on the preoperative CT results, were referred to. The screw insertion process is described in detail in The point located at the distal tip of the curved pedicle probe that entered the LMD was designated the MPPP in this study. Based on this point, the lateral aspect of the lateral mass was not decorticated to create a funnel-shaped hole ,15, the The pedicle probe was then inserted to a depth 2 mm less than the pedicle total length (PTL), directed at an angle corresponding with the appropriate horizontal and pedicle insertion angles (PIA). As an optional safety measure, anteroposterior and lateral radiographs could be taken at this point using an image intensifier to confirm the direction of the probe, as well as to guide it to the correct horizontal angle, following the direction of the pedicles on lateral X-ray or sagittal CT scans in complex cases. After forming a track with the curved probe, ball-tip probe palpation was performed to confirm the integrity of the pedicle borders. A 2.5 mm diameter tap was used to make screw insertion easier and lessen the torque required to insert the screw. Finally, a ball-tipped probe was used to confirm the integrity of the pedicle borders after tapping; if the integrity of the pedicle wall was not clearly confirmed using the ball-tipped probe or if bleeding occurred from the hole where the tapping was performed, it was judged that the integrity of the pedicle wall was broken. In this case, coagulation was performed using bone wax in the concerned hole, and a lateral mass screw was used as an alternative. A 3.5 mm pedicle screw of appropriate length  was then inserted following the same horizontal angle and PIA. After screw insertion, cervical spine anteroposterior and lateral radiography were performed to determine whether the inserted screw trajectory was appropriate .Postoperatively, anteroposterior and lateral radiographs , as wellStatistical analysisp < 0.05. All statistical analyses were performed using SPSS software, version 22 .The means and corresponding standard deviations were calculated for all linear and angular pedicle parameters, while the intraclass correlation coefficient (ICC) was analyzed to determine the perforation grade. Forward stepwise multiple linear regression analysis was used to identify potential risk factors for pedicle perforation, including age, sex, vertebral level, PD, LMD, FPD, and PIA; statistical significance was set at p = not significant, t-test). Most patients  underwent three- to five-level operations. The underlying diagnoses were cervical stenosis , ossified posterior longitudinal ligament with cervical myelopathy , and multiple-level herniated cervical disc disease . The mean duration of surgery—including decompressive laminectomy and/or additional foraminotomy at the designated cervical spine levels—was 205.7 ± 23.5 (range: 122–341) min; the mean blood loss was 362.9 ± 54.6 (range: 100–1300) mL. Preoperative radiographic parameters, including PD, PIA, LMD, FPD, and PTL, are shown in This study included 38 (66.6%) males and 19 (33.4%) females, with mean ages of 63.9 ± 12.29 years and 63.94 ± 11.47 years, respectively (p < 0.001). Perforations of Grade 1 or higher occurred with 54 pedicle screws, representing an overall perforation rate of 19.9% (54/271). Most perforations (80.1%) among all cervical levels were classified as Grade 0 (no perforation), followed by Grade 1 (16.2%) and Grade 2 (3.7%); Grade 3 perforations were not observed , indicating that the likelihood of perforation increases with increasing PIA.Forward stepwise multiple linear regression analyses were also used to identify potential risk factors—including age, sex, vertebral level, PD, LMD, FPD, and PIA—for the occurrence of perforation (Grade 1 or higher). PIA was positively associated with the presence of perforation ; the remaining perforations were Grade 2 (3.7%). Since the screw diameter was 3.5 mm, following the strict surgical indication of a PD ≥3.5 mm was important for our technique. Interestingly, most previously reported techniques did not consider PD when planning surgery, which may have resulted in a higher rate of pedicle breach or pedicle fractures when the screws were inserted ,19,22,24As revealed in the stepwise multiple regression analysis for the occurrence of perforation in this study, PIA was a risk factor. Cervical pedicle placement using the MPPP can therefore be established as a safe technique for lateral wall perforation; however, when the PIA is very high, it can be perforated through a relatively thick medial wall. Nevertheless, even if the medial wall is perforated by purchasing a pedicle screw centered on the MPPP while using the lateral mass as a buttress, the pedicle screw would have already progressed to the cortico-cancellous core, resulting in minimal violation. Accordingly, nerve root injury should rarely occur with the use of our technique, since the dorsal and ventral nerve roots are located in the inferior portion of the neural foramen, both at and below the disc level . ContrasOur freehand technique for cervical pedicle screw fixation using the MPPP may be safe and accurate in patients with a PD ≥ 3.5 mm. Linear and angular measurements on preoperative CT images for each cervical pedicle can be used as a simple and accurate guide when the PD is ≥3.5 mm.


Ice caves constitute the newly investigated frozen and secluded model habitats for evaluating the resilience of ice-entrapped microbiomes in response to climate changes. This survey identified the total and active prokaryotic and eukaryotic communities from millennium-old ice accumulated in Scarisoara cave (Romania) using Illumina shotgun sequencing of the ribosomal RNA (rRNA) and messenger RNA (mRNA)-based functional analysis of the metatranscriptome. Also, the response of active microbiome to heat shock treatment mimicking the environmental shift during ice melting was evaluated at both the taxonomic and metabolic levels. The putatively active microbial community was dominated by bacterial taxa belonging to Proteobacteria and Bacteroidetes, which are highly resilient to thermal variations, while the scarcely present archaea belonging to Methanomicrobia was majorly affected by heat shock. Among eukaryotes, the fungal rRNA community was shared between the resilient Chytridiomycota and Blastocladiomycota, and the more sensitive Ascomycota and Basidiomycota taxa. A complex microeukaryotic community highly represented by Tardigrada and Rotifera (Metazoa), Ciliophora and Cercozoa (Protozoa), and Chlorophyta (Plantae) was evidenced for the first time in this habitat. This community showed a quick reaction to heat shock, followed by a partial recovery after prolonged incubation at 4°C due to possible predation processes on the prokaryotic cluster. Analysis of mRNA differential gene expression revealed the presence of an active microbiome in the perennial ice from the Scarisoara cave and associated molecular mechanisms for coping with temperature variations by the upregulation of genes involved in enzyme recovery, energy storage, carbon and nitrogen regulation, and cell motility. This first report on the active microbiome embedded in perennial ice from caves and its response to temperature stress provided a glimpse into the impact of glaciers melting and the resilience mechanisms in this habitat, contributing to the knowledge on the functional role of active microbes in frozen environments and their response to climatic changes. Ice can be considered as a storage matrix for microorganisms, representing a source of genomic diversity and a reservoir of new microbial species . RecentlThe Scarisoara ice cave (Romania) harbors the oldest and largest perennial ice block accumulated in a cave worldwide , represeThese reports based on DNA sequencing provided information on the total communities, while no data on the metabolically active microbiome from this habitat have been provided so far. Recently, the total and potentially active bacterial communities from a 13,000-year-old ice core from Scarisoara have been determined using 16S ribosomal RNA (rRNA) Illumina sequencing , suggestAlthough reports on metatranscriptomes from frozen habitats revealed the presence of active microorganisms in ice , the occIn this context, the current survey represents the first investigation unraveling active prokaryotic and eukaryotic microbial communities in millennium-old underground perennial ice accumulated from caves based on total RNA [rRNA and messenger RAN (mRNA)] Illumina sequencing. Moreover, our data report the changes occurring at the taxonomic and metabolic levels in the total and active microbial communities in response to a 3-day cycle treatment followed by incubation at 4°C up to 14 days, which were done in order to understand the impact of glacier melting on the ice-embedded microbiome.14C-dated to more than 10,500 years before present (BP) (The Scarisoara ice cave located in the Apuseni Mountains (NW Romania)  harbors ent (BP) . The parent (BP)  ensure uent (BP) . The accent (BP) .Previously , ice samThe combined cave ice core samples were transferred in a 20-L autoclaved glass bottle under aseptic conditions using a microbiological biosafety cabinet to avoid contamination. After slow thawing at 4°C, the melted ice was equally distributed (1.7 L per sample) in 12 sterile 5-L bottles and submitted to thermal treatment . The pro® PowerSoil ® Total RNA Kit  according to the manufacturer’s protocol. DNA contaminants were removed from the resulted RNA (50 μl) with the DNeasy Max ® Kit , and the purity and yield were determined by fluorometry using a Qubit 2.0  in the presence of a Qubit DNA HS Assay Kit  and Qubit RNA HS Assay Kit, respectively. The RNA integrity number (RIN) was determined using a Bioanalyzer 2100 .The protocol for the preparation of RNA extraction and complementary DNA (cDNA) Illumina shotgun libraries  was adap® Ultra II Directional RNA Library Prep Kit for Illumina ® . The subsequent indexing process was carried out using the NEBNext Multiplex Oligos for Illumina and Index Primers Set 1 and the final cDNA library then purified using Sample Purification Beads . All the reactions were performed according to the manufacturer’s protocol in a dedicated PCR clean room. The cDNA library was quantified using a Qubit 2.0 spectrophotometer  and Qubit DNA HS Assay Kit . Library quantification was carried out using the KAPA Biosystems Library Quantification Kit for Illumina ® Platforms  performing a series of 10-fold dilutions according to the manufacturer’s instructions. The final library quality control (QC) and the fragment size distribution were assessed using an Agilent Bioanalyzer 2100 and DNA Chips .To perform the Illumina shotgun sequencing, a specific cDNA library was prepared for each of the 12 samples using 100 ng RNA and the NEBNext The resulting equimolar metatranscriptomic libraries obtained from each T0, T3, T7, and T14 triplicate were pooled and sequenced (150 bp paired-end) using a NextSeq 500/550 high-throughput kit v2.5 and the Illumina NextSeq platform  at the Department of Environmental Sciences, Aarhus University, Denmark.Data Availability Statement for access code) were processed to assess the bioinformatics quality control and downstream analysis. Adapters and reads with an average quality score less than q20 and shorter than 60 bp were filtered using fastp  to adjust for type 1 error.To identify the genes from eggNOG, CAZy, and NCycDB, gene families or functional subsystems that were significantly differentially expressed at different heat shock times, the DESeq2  module oIllumina shotgun sequencing of the total rRNA extracted from the untreated and thermal-treated cave ice microbiome generated a total of 414,992,189 paired-end reads assigned to potentially active bacterial, archaeal, and eukaryotic taxa.Taxonomic assignment of the putatively active microbiome from the cave ice deposit  led to tThe initial cave ice microbial community (T0) was dominated by potentially active Bacteria (95.66 ± 0.76%), with a minor presence of Archaea (0.18 ± 0.02%) and low relative abundance of Eukarya (4.16 ± 0.43%) representatives .Application of the heat shock cycles (T3) had a strong impact on the composition of the ice microbiome, leading to 9- and 8-fold decreases of the relative abundance of Archaea (0.02%) and eukaryotes (0.53 ± 0.02%), respectively, while the bacterial community appeared more stable prevailing (99.45 ± 0.02%) in the ice community exposed to temperature fluctuations . Among tFurther incubation at 4°C for 2 weeks led to a partial recovery of the eukaryotic community, with relative content increases of up to 1.40 ± 0.72% (T7) and 5.49 ± 1.17% (T14) . This trOverall, the response of the ice microbiome to heat shock treatment indicated a high resilience and a faster recovery of the bacterial taxon compared to the archaeal and eukaryotic taxa .Microbial composition at the phylum level of the untreated (T0) cave ice potentially active (rRNA) bacteria indicated the presence of 53% Proteobacteria, 17.4% Chlorobi, 10.8% Actinobacteria, 8% Bacteroidetes, and 5% Firmicutes . The higAt the class level, Proteobacteria was mostly represented by Betaproteobacteria (68%), also containing Alphaproteobacteria (18%), Gammaproteobacteria (10%), and Deltaproteobacteria (3%) prior to heat shock exposure (T0) . Within Cytophagia after 14 days (T14), indicating the high resilience of Flavobacteriia phylotypes to thermal stress  and Chlorobiaceae genus 2 (28%) were the dominant ones, suggesting an important autotrophic activity. The initial microbiome structure also revealed an important (5–8%) presence of the genera Polymorphobacter, Clostridium sensu stricto, Pseudomonas, Carnobacterium, Unknown Chlorobiaceae genus 1, Cryobacterium, Polaromonas, and Flavobacterium. Heat shock treatment induced a redistribution of the major bacterial genera in the T3, T7, and T14 samples, leading to relative content increases from 6 to 15% (T7) and 20% (T14) of Polaromonas spp., a psychrophilic genus ubiquitous in glacial systems .The putatively active archaeal phylotypes identified in the cave ice samples were assigned to class Methanomicrobia (Euryarchaeota) and showed a strong drop in relative abundance after thermal treatment, from 0.16 ± 0.01% (T0) to 0.02 ± 0.00% (T3) and 0.01 ± 0.00% in both T7 and T14 samples. Other archaeal reads were identified only at This rRNA-based survey constituting the first report on the potentially active fungal community structure in ice caves showed the presence of a complex community representing 0.23% of the Scarisoara ice microbiome  assignedAt the phylum level , the iniAt the class level , the iniThe high relative abundance of Chytridiomycetes could be associated with the wide spread of this phylum in freshwater, marine habitats, and in soil , accordiRhizophydium (Chytridiomycota), Geoglossum (Ascomycota), and Ochroconis (Ascomycota) taxa, with relatively similar representation. Rhizophydium species belonging to Rhysophydiales are parasites of invertebrates, chytrids, and algae, assuming a possible role in the control of aquatic populations, and are also common in soil, primarily as saprobes, with a possible role in nutrient recycling  . After tAnalysis of the taxonomic distribution of the microeukaryotes in the untreated T0 microbiome  revealedHeat shock treatment (T3) reduced the relative content of Chlorophyta (5%) and increased those of the taxa belonging to Metazoa (15%), Ciliophora (30%), Cercozoa (10%), and Bicosoecida (12%). After a week of incubation at 4°C (T7), the microeukaryotic community was dominated by Bicosoecida (80%), with a severe representation loss in the Metazoa group (1%). The prolonged incubation (T14) of ice samples induced further changes in the distribution of microeukaryotes, leading to a clear dominance of Chrysophyceae (62%) and a reduction of the relative content of Bicosoecida to 20% .Potentially active Protozoa community from the 900-O untreated (T0) cave ice samples occupied 0.8% of the rRNA identified microbiome . AssessmThe Metazoa group from the Scarisoara 900-O ice sample (T0) constituted 0.26% of the potentially active (rRNA) cave ice microbiome , mainly Oikomonas, Spumella, and Bicosoeca, displayed an increase in T7, with the highest representation after 14 days. The very high abundance of these genera could be explained considering a combination of the characteristics of Stramenopiles and the decline of the relative contents of prokaryotes in this sample. Stramenopiles are characterized by a smaller cell size compared to other protozoa  was modeled by both the increased temperature and prolonged incubation at 4°C, with the subsequent activation of different processes corroborated by the upregulation of specific gene clusters .Functional annotation using the eggNOG database revealed 639 differential functions. The impact of thermal shock and prolonged incubation on the metabolism of the active microbial community harbored in the Scarisoara millennium-old ice was deciphered by evaluating the gene expression profile of this microbiome belonging to different groups and after each thermal step .While the T0 sample showed a reduced general transcription, the T3 microbiome revealed the highest increase in mRNA overall transcripts . ThermalAnalysis of the differential gene expression between the thermal treatment steps of various functional genes from the eggNOG database  showed aA pronounced increase in the transcripts associated with the tricarboxylic acid (TCA) cycle  was visible after the thermal shock step (T3), while a distinct reduction was evident after 7 and 14 days of incubation . The incTranscripts involved in amino acid transport  and related to carbon and nitrogen metabolism using amino acids as intermediates  were also upregulated after the application of thermal treatment . The traEscherichia coli, indicated a direct correlation with the production of reactive oxygen species (ROS) in response to heat treatment . Although the level of GroEL transcripts increased right after the ice thawing (T0), a significant decrease of this gene expression was observed , which sreatment . Furtherreatment , was higreatment , suggestIn addition, prolonged incubation for 14 days resulted in an increase in the defense mechanisms  gene expression , suggest2 emissions as a primary driver of climate change.Elevated temperatures also accelerated fungal decomposition, resulting in increased carbon dioxide emission from the soil, which can lead to a faster temperature rise. A more detailed analysis could help in identifying the differential expression of genes involved in microbial respiration. Therefore, these genes could also be considered as potential markers for identifying specific microbial taxa adapted to higher temperatures that could contribute to global COAlthough development of the investigated system in the long run remains an open question, the short-term temperature changes  appeared to shape the active microbial community embedded in the Scarisoara cave ice. Extended investigations on long-term temperature variations will help deepen the knowledge on the microbial role in the global climate change.To date, this study provides the first evidence of an active microbiome and putatively active microeukaryotic taxa in perennial ice from caves, in addition to initial data on the thermal treatment response of the total and active ice-entrapped microbiomes using Illumina shotgun sequencing of rRNA and mRNA shift analysis. Temperature changes are known to disturb the microbial homeostasis alongside an altered taxon distribution, while little is known about the transcriptomic response to thermal stress. This overview of the structural and functional shifts in the ice microbial community induced by heat shock cycles and prolonged incubation contributed to increasing our understanding on changing environments and their ecological impacts due to ice melting.The taxonomic profile of the potentially active microbiome from this icy environment was primarily modeled by heat shock, meanwhile revealing resilience mechanisms and specific functional responses to thermal stress. The ice community dominated by copiotrophic taxa was able to quickly use the carbon source released after ice thawing, with the Proteobacteria and Bacteroidetes taxa prevailing after thermal stress, unlike Archaea showing no recovery after incubation at a low temperature and the highly affected fungal community structure. Meanwhile, microeukaryotes exhibited fast recovery based on the putative predation process on bacteria. The resilience strategies of the active ice microbiome after heat shock exposure involved refolding, oxidative stress, and DNA synthesis-related gene upregulation. An increase in energy production and storage occurred during thermal stress, in accordance with the distribution of the dominant bacterial classes endowed with a copiotrophic lifestyle. Boosted carbon and nitrogen regulation and cellular motility appeared to respond to requirements for the water environment after ice melting, while the activation of defense mechanisms suggested a start of competition for similar food sources. The reported structural and functional modifications of the active microbial community from the Scarisoara cave ice due to thermal variations offered a glimpse of the environmental impacts of climate change leading to glacier retreat. In-depth analyses of the reconstructed metagenomes could contribute to unraveling the evolutionary patterns of metabolic pathways in this secluded cold habitat, subtle molecular adaptation mechanisms to icy environments, and novel cold-active biomolecules of applicative potential. Meanwhile, this first report on a critical shift of the active microbiomes from melting ice habitats represents a warning for possible major changes of the environmental biogeochemistry associated with late accelerated ice loss.PRJNA777283 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA777283).The sequencing datasets presented in this study are publicly available at Sequence Read Archive (SRA) under the BioProject ID AM and CP wrote the manuscript. AM performed the sample filtration, heat shock experiment, RNA extraction, and cDNA library preparation. LE-J and CJ designed the thermal treatment experiment and carried out the Illumina shotgun sequencing with contributions from AM and MZA. MZA performed the bioinformatics and statistical analyses. AM and PL contributed to the sequence analyses. CP performed the experimental design and coordinated the project. All authors contributed to data interpretation and revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.