Among the obtained 30,846,354 transcripts sequenced, 30,303 differentially expressed genes from the control and stress treatment samples were identified, with 3363 genes expressed ≥ 2 and false discovery rate (FDR) of < 0.001. Six differential expression genes were then selected and qRT-PCR was used to confirm the RNA-seq results. Some of the genes  and signaling pathways discussed in this paper not been previously studied in quinoa. Genes with ≥ 2 were used to design the gene interaction network using Cytoscape software, and AgriGO software and STRING database were used for gene ontology. The results led to the identification of 14 key genes involved in salt stress. The most effective hub genes involved in salt tolerance were the heat shock protein gene family. The transcription factors that showed a significant increase in expression under stress conditions mainly belonged to the WRKY, bZIP and MYB families. Ontology analysis of salt stress-responsive genes and hub genes revealed that metabolic pathways, binding, cellular processes and cellular anatomical entity are among the most effective processes involved in salt stress.To better understand the mechanisms involved in salinity stress, the adaptability of quinoa cv. Titicaca—a halophytic plant—was investigated at the transcriptome level under saline and non-saline conditions. RNA-sequencing analysis of leaf tissue at the four-leaf stage by Illumina paired—end method was used to compare salt stress treatment (four days after stress at 13.8 dsm Halophytes are one of the best germplasms in terms of Reactive oxygen species (ROS) detoxification and signaling2. Most metabolic pathways lead to the continuous production of ROS, which negatively affects biological molecules1. Quinoa  is a facultative halophyte native to the Andes in Bolivia and Peru and is considered as an alternative to major crops to eliminate the present food shortages3. This plant has a strong root system and is highly resistant to a wide range of abiotic stresses3. Some varieties of quinoa can even grow at salinity concentrations higher than seawater4. Plant adaptation to salinity stress through anatomical and physiological changes resulting from primary salt stress signaling pathways of salinity stress such as ROS, Ca+2 diffusion and phospholipid signaling6. Despite extensive information on the mechanisms of salinity tolerance in quinoa, studies on this topic are still limited to the transcriptional level7.Salinity is recognized as one of the most important environmental limitation+2 signaling is received by various proteins such as calcineurin B-like protein (CBL), Calmodulin (CaM), calmodulin-like proteins (CML) and calcineurin B-like protein-interacting protein kinase (CIPK), which in turn regulate the downstream targets and consequently, release the signaling cascade9. Analysis of the gene expression profile analysis showed that CMLs play a key role in the response to abiotic stresses such as drought and salinity11. Until now, CaMs and CBLs are the sole representatives of sensor relay proteins which transduce the signals via molecular interactions after binding to the second Ca+2 messenger12. It is suggested that Cystathionine b-synthase (CBS) proteins maintain cell redox homeostasis and regulate plant growth with the help of Thioredoxin (TRX) systems directly in Ferredoxin-Trx system (FTS) and NADP-TRX system (NTS), which consequently leads to the control of H2O2 levels13. glutaredoxins (GRXs)are placed into TRX superfamily along with thioredoxins. They are members of a multigene family of proteins and are considered as maintenance and regulatory mechanisms15. Maintenance of redox balance in the cell is critical for various signaling pathways and metabolic activities and is done by different isoforms of GRX14. Some protein kinases in the cell can sense the production or induction of ROS and respond to the stress via a series of phosphorylation and dephosphorylation signals. For example, NADPH oxidase can rapidly increase intracellular ROS levels and the induced signal is received by the nucleus through the plasma membrane16. Two protein kinases (SnRK2.4 and SnRK2.10) are released by SNF-related Kinase 1 (SNF1), the key component of cell cellular signaling network, to regulate ROS homeostasis and response to salinity stress in Arabidopsis17.Ca+2 and acts as a mediating molecule to bind HSP70/HSC70 to the target protein18. Results of qRT-PCR analyses showed that AtBAG6 transcription level was significantly upregulated by abiotic stresses such as salinity20. Also, three  out of seven BAG proteins identified in arabidopsis have distinctive properties unique to plant BAG proteins which are probably regulated by calmodulin and Ca+221. BAG family proteins are involved in various cell processes such as apoptosis, proliferation, differentiation and stress signaling21. Most molecular chaperones are stress proteins that exist as HSPs that strongly protect the cell against from injury such as salinity stress22. For example, the expression of 9 genes of HSP family increased under salinity stress in rice23.Calcium signaling plays directly regulates programmed cell death (PCD) by protein folding through chaperones. Bcl-2-associated athanogene (BAG) protein includes a BAG domain which binds to heat shock cognate protein 70 (HSC70) and a specific IQ motif which binds to the free cytosolic Ca24. Seawater is a mixture of saline solutions, similar to saline soils, and their synergism can affect seed germination25. Due to the potential of quinoa to grow under adverse conditions, sowing this plant in regions with saline water sources may be a good option.Most studies on the effects of salinity on halophytes have been conducted with NaCl.This approach does not provide comprehensive information about the tolerance potential under field conditions, because the soil contains different salts that affect growth and germination26. This method can help to investigate the identified candidate genes identified from transcriptomic studies in a molecular interaction network. In this view, the clusters of this interaction network with the highest relationship between the candidate genes are identified as the major biological processes involved in a given study. This issue makes it possible to have a more holistic view about the studied process26. Quinoa is a potential crop in the middle east, northern Africa and central Asia, which have saline soil and water and thus struggle with limitations in crop production. To fully exploit the potential of quinoa as a suitable crop in marginal environments, identification and introduction of new high-yielding genotypes and by finding key genes or introduction of genes to current high-yielding cultivars is necessary, and our paper may help in this regard. The aim of this study was to use RNA sequencing and network analysis in the quinoa plant to investigate the genes involved in dealing with salt stress management and also to identify the effective hub genes, with the help of RNA sequencing and network analysis in quinoa plant.The development of high-throughput sequencing technologies has allowed researchers to use the RNA-seq method to identify and compare the pattern of genes that affect salt tolerance and ultimately to describe the molecular mechanisms of tolerance in plants. Network analysis is an effective method for combining experimental data obtained from different molecular levels with all available molecular data−1 and 13.8 dsm−1, 4d treatment at 13.8 dsm−1 was selected along with the control plant.According to the results of biochemical and molecular analyses such as superoxide dismutase, peroxidase, polyphenol oxidase, catalase, proline, glycin-betaine (data not shown), among the treatments 6 h, 1d, 2d, 3d, 4d, 5d, 6d and 7d after salinity stress at 6.9 dsmResponse to salinity stress in quinoa plant cv. Titicaca was investigated using the RNA-seq technique. Among the total reads, 31,676,929 transcripts for control and 30,800,872 transcripts for 4 days after salinity stress treatment were specifically mapped to the reference genome using Star Aligner. The average length of the mapped reads was 197 bp , mitochondria (CBSX3), cytosol (CBSX4) and endoplasmic reticulum (CBSX5 and CBSX6) and it can be safely assumed that CBSX is required for a sensor relay protein such as CaM and CBL13. Furthermore, proteins possessing the CBS domain are found in all kingdoms of life except for viruses, thus far. For instance, the number of proteins containing the CBS domain identified in E. coli, Saccharomyces cerevisiae, Arabidopsis Thaliana, Oryza Sativa and Homo Sapiens is 8, 12, 34, 59 and 75, respectively40. Results of RNA-seq led to the identification of 9 proteins that contained the CBS domain in quinoa. Pathways obtained from the STRING database also indicated the activity of this gene in the metabolic pathways.In plants, ss Figs. c and 6c.nt Figs. c and 6c.GRX gene to Arabidopsis resulted in improved cold tolerance in the plant41. overexpression of the GRX gene under heat (45 °C), cold (4 °C) and saline (150 mM NaCl) conditions also indicated tolerance in Oryza Sativa42. Investigation of gene expression profile in Arabidopsis in response to various biotic and abiotic stresses showed that the GRX and TRX genes play a key role in stress tolerance43. The number of TRX genes in plant species may vary from 11 in sorghum to 60–70 in rice and Arabidopsis44. In the present study, RNA-seq results revealed that the total number of TRXs and GRXs identified in quinoa was 19 and 51, respectively. It seems that cold stress decreases the expression of most TRX genes, but drought stress -at least in the early stages of stress- leads to the upregulation of this gene45. Since GRXs are members of the TRX family, the interaction of these genes leads to upregulation of CBSX5 and helps to improve stress tolerance by maintaining the balance and control of H2O2 in the cell. Previous studies have shown the critical role of GRXs in the tolerance to abiotic stresses such as oxidative stress and metals46. In a study conducted by Kumar et al.47 on two cultivars of chickpea (Cicer arietinum L.), the CaGRX gene was investigated for overexpression and various biochemical and physiological parameters related to salinity and drought stresses. The results showed that CaGrx improved the plant tolerance to drought and salinity by positive regulation of the antioxidant defense system and different stress-related parameters. Increased CaGrx expression improve plant biochemical and physiological performance in response to drought and salinity stress by activating the antioxidant defense system. The decrease in ROS levels under high salinity and drought conditions may be due to the overexpression of CaGrx, which increases catalase and APX activity and directly decreases H2O2 levels and DHA expression48, which is in accordance with the results of the present study. As a defense mechanism, GRX limits excessive ROS production, participates in redox signaling, and directly or indirectly enhances antioxidant defense mechanisms47. Gene ontology of the TRX1 and GRXC9 genes showed that these genes are related to the process of organic substance metabolism, cell process single organism process and biological regulation, involved in the biological processes.In living cells, ROS play a key role in signal transduction. However, these compounds also damage the macromolecules. The concentration of ROS in mitochondria- as well as in other compartments- must be strictly controlled. Plant mitochondria contains several antioxidant systems that can repair the damage to macromolecules and probably act as redox sensors. These include glutathione-dependent pathways and systems based on glutaredoxin (GRX) and thioredoxin-like (TRX) molecules. In one experiment, the transfering of the 49. Previous studies have shown that TFs are activated simultaneously with protein kinases, which act as signal transmitter/receiver proteins in the membrane50. These TFs included the bZIP, C2H2, BHLHs, ERF, MYB, NAC and WRKY families and each had a different expression in salinity tolerance in plants, which is in accordance with the results of Arisha et al.50 According to the RNA-seq results in the present study. All of these TFs had specific and significant expressions 4 days after salinity stress treatment, which indicates the mechanisms of stress tolerance (Table −1 in the last days of stress. These genes were slowly activated within hours after stress and often showed a stable levels of expression. Transcription factors such as early genes activate and encode the most important genes that respond to stress (delayed genes).The SnRKs family is divided into SnRK1, SnRK2 and SnRK3 subfamilies. It is established that most members of the SnRKs family play essential roles in response to abiotic stresses. SnRKs cooperate with TFs in the maintenance of cellular energy balance17 found out that SnRK2.4 and SnRK2.10 in cooperation with SnRK1 can maintain the ROS homeostasis and response to salinity stress in Arabidopsis17. SnRK1 acts as a key kinase in stress response, and overexpression of PpSnRK1α can significantly improve salinity tolerance by regulating ROS metabolism regulation or ABA-mediated pathways. It has been reported that the overexpression of the gene encoding the α-subunit of SnRK1 in Prunus persica (PpSnRK1α) can enhance salinity tolerance. Overexpression of SnRKγ1 led to lower leaf damage, increased proline, and decreased malondialdehyde (MDA) content compared with the control under salinity stress conditions (data now shown), which was similar to the results of49. The results of qRT-PCR for the SnRKγ1 gene showed that the expression of the gene in the plant increased during the days after the stress. A significant increase at 6.9 dsm−1 began on the 3rd day and peaked after 4 days of exposure. In fact, when the plant is affected by ROSs, the expression of this gene at high levels neutralizes the effects of ROSs and prevents the induction of stress7. This also occurred at 13.8 dsm−1 salinity level, and since the plant had to cope with more salt content at this level, increased expression started from the 1st day and continued until the end. It may be concluded that the plant utilized the mechanism of increased SnRKγ1 expression to prevent the excessive consumption of ATP as well as to control the adverse effects of ROSs.Szymańska et al.BAGs also regulate HSP chaperone proteins (positively or negatively) and form complexes with various transcription factors51. At the transcriptional level, BAG family genes in plants have key roles in the PCD processes which range from growth, and tolerance to fungi to abiotic stress tolerance52. The results of the expression of this gene at 6.9 dsm−1 salinity level showed that from the early days of stress, the plant increased the expression of the BAG6 gene to prevent the stress, so that on the 2nd day, cell apoptosis and PCD were prevented by overexpression and with the help of chaperones. This reached it is peak 4 days after stress in 13.8 dsm−1 treatment. It seems that the damage inflicted upon the plant has irreversible effects which leads to high energy consumption. However, the cooperation between the BAG family and chaperones will help the plant to maintain its balance under adverse conditions. Under abiotic stress, a strong induction of the genes in the BAG family has been observed21. Also, it is demonstrated that ABA is involved in the regulation of BAG gene in Arabidopsis thaliana and plays a critical role in response to abiotic stresses21. GO results for the BAG6 gene showed that this gene is associated with chaperone binding and protein binding category and most interact with chaperones and binding proteins to mitigate the stress through a significant increase in the expression level  in protoplasts of transgenic rice affected the PCD62. HSP70 interacts with the members of the Bcl-2 family and prevents cell apoptosis64. HSP70 is usually required for polypeptide movement across the mitochondria inner membrane and further reactions of protein folding in the matrix65. The redox conditions of thiol-containing molecules (TRXs and GRXs) are important for cell performances such as synthesis, folding and structural regulation of proteins and transcription factors62.According to the results, the endoplasmic reticulum pathway was observed along with the increased expression of udy Fig. , rank 6.These results indicate that an important part of the salt tolerance mechanism cannot be determined using GO analysis. To better understand the mechanisms of salt tolerance mediated by candidate genes, network analysis was utilized and sub-networks involving a large number of candidate genes and lowest edges were identified. The advantage of this method is that all molecular information available on quinoa as well as information obtained from other gene expression studies, can be utilized in an interactive network to extract more useful results and comperhensive understanding.−1 (HSPs family). These genes are expected to be essential in salt tolerance and it may be concluded that they can increase the tolerance threshold of quinoa to salt stress either individually or together. Among these genes, the effective role of WRKYs, bZIPs and MYBs may also be mentioned. Ontology analysis of the genes responding to salinity and hub genes revealed that protein processing in the endoplasmic reticulum is an important pathway involved in this stress. The protective effect of HSPs/chaperones may be attributed to the chaperone mechanism network in which many chaperones act in a coordinated manner. Under stress, many structural proteins are subjected to negative structural and functional changes. Therefore, refolding of the denatured proteins and maintenance of their function is critical for the survival of cells under stress conditions. These findings may be promising to update our knowledge about the role and changes in the genes involved in salt tolerance. This knowledge may be applied to the cultivation of halophytic plants such as quinoa using non-conventional water sources.In the present study, first, the genes responding to salinity stress in quinoa were identified and some were further analyzed for the first time. The assembled transcript was used to investigate differential expression and annotation of genes. We identified 3363 genes with differential expressions based on FDR < 0.001 and FC ≥ 2. The differential expression pattern for six of these genes was confirmed by qRT-PCR analysis and each demonstrated a similar level of up- or down- regulation. In the second part of the study, the reconstruction of the network of genes and the interaction of related proteins led to the identification of hub genes at 13.8 dsmAt all stages, the research complied with relevant institutional, national, and international guidelines and legislation.67 was purchased from the Iranian National Salinity Research Center, Yazd, Iran. Salinity stress at 6.9 dSm−1 (1:1 sea water and double distilled water), 13.8 dSm−1 (sea water) and control (double distilled water) were applied with four biological replicates. The seawater originated from the Caspian Sea. Each pot contained loamy soil and a mixture of sand and humus (2:1). Salinity treatments were applied at the end of two leaf stage. The leaves were sampled at the end of four-leaf stage (30 days after emergence) 6 h, 1, 2, 3, 4, 5, 6 and 7 days after treatment. The samples were then immediately frozen in liquid nitrogen and kept at −80 °C.To investigate the transcriptome of quinoa under salinity stress, Titicaca genotype which is early-maturing and tolerant to adverse environmental conditions such as drought, cold and salinityDNase I enzyme. Quantity and quality of the extracted RNA was confirmed using spectrophotometry at 260 nm and 1.5% agarose gel. Since parameters related to the enzymes and biochemical attributes (data not presented in the paper) were higher 4 days after treatment with 13.8 dSm−1, this treatment was selected along with the control to investigate the profile of total transcripts. This was done at BGI company  using RNA-seq and NextFlex kit. Construction of cDNA library was done on 2500 Illumina Hi seq ™ 2500  platform. Two cDNA libraries were constructed from the mRNA extracted from the control and 4 days after treatment (13.8 dSm−1) leaves of quinoa. Measurement with the Bioanalyzer instrument showed that all samples had RIN values of > 7.5 and therefore suitable for the construction of cDNA library and sequencing.Total RNA was extracted using p-BIOZOL kit  from the samples and then treated with 68. The sequence of Chenopodium quinoa transcript was downloaded from https://plants.ensembl.org/Chenopodium_quinoa/Info/Index. Then, the filtered reads along with the quinoa genome as the reference and Gene annotation were entered into STAR (v 2.7) software. High quality reads were mapped on the reference quinoa genome and transcriptomes were assembled. The role of genes were identified on Ensembled Plants (https://plants.ensembl.org/index.html) database. Function of novel genes was investigated using NCBI non-redundant (NR) database and BLASTp software. Identification of differential expression genes (DEG) in the samples was done using the R (v 4.1.2) software (https://www.rstudio.com/tags/website/). For this purpose, after the normalization of expression using edgeR package with trimmed mean of M-values normalization (TMM) method, Log2 value of Fold Change index was obtained for each gene. DEGs with FC values of ≥ 2 and with FDR of < 0.001 were considered as significant. Gene ontology (GO) was used to categorize the expressed functional DEGs. To categorize the genes according to their molecular role, cellular compartment and biological process, the list of GOs of DEGs were analyzed using AgriGO and g:profiler (http://biit.cs.ut.ee/gprofiler/) online software. Pathway enrichment analysis was done using the (https://string-db.org/cgi/input?sessionId=bOY6Uufuj0j2&input_page_active_form=multiple_identifiers). database. Important pathways were selected using Fisher’s exact test at and FDR of < 0.001.Raw data underwent quality control and were edited using FastQC and Trimmomatic software. Reads with adapter sequences were omitted. Also, reads with low quality or unknown bases of > 5% were filtered to obtain high quality readshttps://www.primer3plus.com/) based on the three prime untranslated (′3- UTR) regions. The names and sequences of primer used for real-time PCR amplification are listed in Table −ΔΔCT using REST software69 method. Validation of the results of qRT-PCR and RNA-seq was then estimated using R software. Excel software was used to generate the related figures.To confirm RNA-seq results, 6 DEGs were selected and primers were designed using Primer3 software  in the network, four Cyto-Hubba calculation algorithms  were used.To draw the gene network and find hub genes among DEGs with FC > 2, orthologue genes of quinoa in 27. This algorithm categorizes the clusters regarding the protein complex and is more operational than the other algorithms in this App27. GO pathway was extracted from each cluster using the STRING database.Information from gene ontology and pathways of DEGs with FC > 2 and hub genes  were extracted using Agri GO software and STRING database. The, Cytocluster plugin was then used for better clustering of the subnetworks obtained from Cytohubba plugin


These detectors are promising for applications in the mid-infrared requiring sub-nanosecond timing, ultra-high gain stability, low dark counts, and high efficiency, such as chemical sensing, LIDAR, dark matter searches, and exoplanet spectroscopy.We developed superconducting nanowire single-photon detectors based on tungsten silicide, which show saturated internal detection efficiency up to a wavelength of 10  I.μm.1 Extension of such performance to the mid-infrared has potential for new applications in astronomy,2 as well as LIDAR,3 dark matter searches,4 and the fundamental study of fast molecular dynamics and chemistry.8 Interest in the mid-infrared stems primarily from the presence of numerous absorption signatures for molecules such as water vapor, carbon dioxide, oxygen and ozone, methane, and nitrous oxide. These molecules not only are important for understanding the evolution of our own planet but may also be signatures of life or indicate the potential habitability of planets outside of our solar system. The spectroscopy of exoplanet atmospheres is a prime example of an application requiring single-photon-counting detectors with ultra-stable gain and high efficiency due to the photon-starved nature of the measurements.10Efficient single-photon counting, with a detection efficiency greater than 50%, has, to date, been achieved only at wavelengths shorter than 2 11 Another common technology, which has been used extensively in astronomy, is the blocked impurity band detector (BIB). These are silicon-based detectors that are not single-photon sensitive but, nevertheless, have demonstrated high quantum yield (>60%) over a broad range of wavelengths spanning from 4 to 28 μm.12 BIB detector arrays are not as common as HgCdTe arrays due to the fact that they operate at a lower temperature of ~10 K.Currently, the most common detector technology in the mid-infrared wavelength range is based on the semiconductor HgCdTe. Small-format HgCdTe avalanche photodiode (APD) arrays have become available, which are single-photon sensitive and can operate at a temperature of 77 K or higher.13 Transition edge sensors (TESs) have also been considered as a detector technology, which may be useful for exoplanet spectroscopy although, to date, there has not been much experimental work in optimizing them for the mid-infrared.14 Both of these technologies operate at extremely low temperatures, typically on the order of 0.1 K.Superconducting single-photon detectors have also shown promise in the mid-infrared and in general have the advantages of significantly lower dark count rates and improved gain stability when compared with semiconductor-based detectors. In particular, arrays of microwave kinetic inductance detectors (MKIDs) have been widely used in astronomy due to their large array formats and are being explored for use in the mid-infrared.15 larger dynamic range, higher count rates compared to MKIDs and TESs and intrinsic dark count rates of less than 1 count/day.4 Arrays as large as 1 kilopixel have also been demonstrated, enabling a wide range of new potential applications.16 Although previous work has focused primarily on telecommunication wavelengths, recently there has been significant interest in using these detectors in the mid-infrared. In particular, the low jitter and high dynamic range make SNSPDs ideal for space-to-ground or fiber-based quantum key distribution and LIDAR.17 Low noise and extremely stable gain make SNSPDs ideal for exoplanet spectroscopy.2Superconducting nanowire single-photon detectors (SNSPDs) are another class of superconducting detector, which can operate at relatively high temperatures compared to MKIDs and TESs, typically between 1 and 4 K. SNSPDs have been used extensively in the quantum optics field due to their high efficiency (98% at 1550 nm),μm. Internal detection efficiency is defined as the probability that a photon absorbed in the nanowire generates an electrical output pulse from the detector. Prior work in the near-infrared has shown that achieving saturated internal detection efficiency is a critical step in making high efficiency devices.18 Our results indicate that it may be possible to achieve high system detection efficiency in the mid-infrared with WSi SNSPDs by optimizing the absorption in the nanowires and the coupling to the active area of the detectors, as has been done at near-infrared wavelengths.Here, we report on SNSPDs based on tungsten silicide (WSi) with saturated internal detection efficiency up to a wavelength of 10 20 NbN has historically been the material of choice for SNSPDs due to its relatively high superconducting transition temperature (8–12 K) allowing operation at 4 K and its fast thermal recovery time allowing operation at high count rates.21 In order to improve the energy sensitivity of the nanowire detectors and extend the photo-response to longer wavelengths, there are several possible approaches.Previous approaches to achieving sensitivity in the mid-infrared were based on SNSPDs fabricated from niobium nitride (NbN) thin films.μm using ultra-narrow 30 nm-wide nanowires.19 In another report, single-photon sensitivity was achieved at a wavelength of 10.6 μm using 40 nm-wide NbN nanowires although the internal detection efficiency was not saturated.20The first approach is to decrease the cross-sectional area of the nanowire, making the film thinner or the nanowire narrower. This results in a higher probability of a hotspot (non-superconducting domain) being generated since the energy per unit area is larger, and thermal conduction along the length of the nanowire is smaller. Using this approach, saturated internal detection efficiency has been demonstrated with NbN SNSPDs up to a wavelength of ~3 22 As the nanowire becomes narrower, edge roughness and constrictions in the width of the nanowire, due to fabrication imperfections, begin to have a larger relative effect and degrade the device performance. The constrictions result in a suppressed switching current, the current at which the critical current density in the wire is exceeded and the superconductor switches to the normal (non-superconducting) state. Since the minimum detectable photon energy depends on the fraction of the depairing current that the detector is biased at,25 it is important to minimize the impact of edge roughness and constrictions. It is expected that the edge roughness is on the order of 5 nm, limited by the electron-beam resist processing, which is good enough to yield nanowires down to 50 nm without significant degradation in performance.The second approach to improving energy sensitivity is to engineer the material to reduce the free carrier density. Reducing the free carrier density (increasing resistivity) of the material results in the deposited energy being divided among fewer quasiparticles, thus increasing their effective temperature. Simultaneously, the thermal impedance along the length of the nanowire is increased, which more effectively localizes the deposited energy along its length. This approach allows the width and thickness of the nanowires to be larger, which is better from a fabrication and yield perspective since the maximum achievable fraction of the depairing current has been shown to decrease for narrower nanowires.26 However, reducing the superconducting gap energy implies a lower superconducting transition temperature (Tc) and thus a lower operating temperature for the detectors. This is generally undesirable as it results in an increase in the complexity and cost of the cryogenics.A third approach to improving energy sensitivity is reducing the superconducting gap energy. This results in a larger number of broken Cooper pairs (quasiparticles) for a given amount of deposited energy in the superconductor and a larger fraction of photon energy accumulated in the electronic system.Tc is not strongly dependent on stoichiometry as shown in 27 as verified by x-ray diffraction measurements for the samples presented here.As outlined above, the most promising approach to lowering the energy threshold is to increase the resistivity of the superconducting film, which is typically achieved through the variation of the stoichiometry. For amorphous WSi, II.Tc value of the thin film is significantly lower than that shown in Tc with film thickness. Each detector is a single 10 μm-long nanowire instead of a large-area meander in order to reduce the probability of fabrication defects or constrictions along the length of the wire for the relatively narrow widths investigated—50 and 70 nm. A 2 μH inductance consisting of a 10 μm-wide meandering wire was patterned into the WSi film in series with the nanowire to prevent latching to the non-superconducting state.28 The scanning electron micrographs of the chip layout are shown in We fabricated detectors from high-resistivity WSi films using a Si-rich stoichiometry. The films are co-sputtered from separate W and Si targets, allowing the composition of the films to be tuned by adjusting the relative sputtering powers. Sputtering is performed with the substrate held at room temperature. Typical sputtering powers for the W and Si targets are 100 and 180 W, respectively, which in our deposition system results in a silicon content of 15 at. %. We increased the Si sputtering power from 180 to 260 W while maintaining the W sputtering power at 100 W. This resulted in a silicon composition of 35% ± 7% as estimated by secondary ion mass spectroscopy (SIMS). The film thickness was 3.2 nm, and the superconducting transition temperature was 3.1 K. Note that the μm) are mounted side by side such that it is possible to study the response to different wavelengths during the same cooldown of the cryostat. The average full-width at half maximum (FWHM) divergence of the 4.8 and 7.4 μm lasers was ∼53° and 60°, respectively, and both QCLs were fabricated at the Jet Propulsion Laboratory (JPL).30 The divergence of the 9.9 μm laser (Alpes Lasers31) was not directly measured but is expected to be similar to the other lasers in the vertical direction and ~40° in the horizontal direction. The QCLs were operated with 260 μs pulses and a 4% duty cycle to minimize the heat load at the 4 K stage. To collect the photon count rate (PCR) curves, a 200 μs gate was applied on a counter, synchronized with the middle of the laser pulses. The number of photons impinging on the detector in a single 260 μs laser pulse was on the order of 100, which ensured that the detector was far from saturation, since its recovery time is >1000 times faster than the duration of the laser pulse. The background count rate (BCR) was collected by synchronizing the gate with the period when the laser is turned off. The signal from the SNSPD was amplified with a cryogenic amplifier (Cosmic Microwave Technology CIT-LF131) operating at the 4 K stage, with a gain of 45 dB, a bandwidth of 1.5 GHz, and a noise temperature of less than 6 K. An additional room temperature amplifier with a gain of 25 dB and a bandwidth of 500 MHz was used, followed by a 120 MHz low-pass filter. This readout scheme enabled pulses to be read out while biased with currents as low as 200 nA.The SNSPDs were measured at a temperature of 0.85 K and flood-illuminated using various quantum cascade lasers (QCLs) also mounted inside the cryostat at the 4 K stage. μm). The presence of a saturated plateau implies unity internal detection efficiency, where each absorbed photon leads to an electrical pulse. As shown in μm photons, which means that it is operating near the saturated internal detection efficiency point. The less-pronounced inflection point  wavelengths (up to 3 μm), it is conceivable to combine narrow-band interference filters together with a sufficient amount of material absorption in glass, such as BK7, to sufficiently reduce the background for single photon applications. For mid-wave infrared (MWIR) and long-wave infrared (LWIR) applications, cryogenic spectrometers5 will likely be required to sufficiently channelize the background.In some applications, the signal to be detected originates from a cryogenic environment, but it should be ensured that the coupling fiber or optics are also cooled,μm wavelength range by adjusting the thicknesses of the germanium layers. With a more complex dielectric layer stack consisting of alternating layers of germanium and a low-index dielectric such as MgF2 or BaF2, the peak efficiency could be increased further, perhaps as high as 80%. Achieving higher than 80% efficiency will be a challenge due to the fill factor, which is limited for fabrication reasons to roughly 50% with these ultra-narrow nanowires.While the initial data presented here appear promising, further work is required to investigate the scalability and yield of narrow wires on the order of 50–80 nm over larger areas, which will be required for kilopixel and larger arrays required for applications such as exoplanet spectroscopy and dark matter science. If larger-area nanowire meanders can be yielded, simulations show that an optical cavity consisting of a gold back-reflector combined with a germanium-based dielectric cavity could yield an efficiency of 55%, comparable to the efficiency of typical blocked impurity band detectors albeit with a significantly smaller bandwidth. The peak wave-length may be tuned within the 2–10 Methods of efficiency calibration of SNSPDs in the mid-infrared are currently being developed at NIST using a cryogenic spectrometer and a reference BIB detector. Both hollow-core optical fibers and chalcogenide glass fibers are well-developed technologies, which can be used for detector coupling.Although we did not measure these SNSPDs at temperatures lower than 0.85 K, we do not anticipate significant improvement in performance with regard to the dark count rate or switching current by operating at lower temperatures. Dark count rates as measured here are limited primarily by the blackbody background. However, one could conceivably improve mid-infrared sensitivity further by decreasing the superconducting transition temperature as discussed in the Introduction. In this case, the use of a dilution refrigerator to achieve a lower operating temperature may be beneficial.Supp1


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that appeared in Wuhan, China in January 2020 and caused a global pandemic drastically changing everyday life. Currently, there are vaccine candidates in clinical trials and development, so it is only a matter of time before one is authorised for human use.We collected public opinion survey results about attitudes towards SARS-CoV-2 vaccination conducted in 2020 in 26 European countries.The pooled surveys were conducted on a total of 24 970 participants; on average only 58% (n=14 365/24 970) of responders across Europe were willing to get a SARS-CoV-2 vaccine once it becomes available, 16% (n=3998/24 970) were neutral, and 26% (n=6607/24 970) were not planning to vaccinate against SARS-CoV-2. Such a low vaccination response could make it exceedingly difficult to reach the herd immunity threshold for SARS-CoV-2 through vaccination.It is very important to start conducting educational public health activities on the topic of vaccination as soon as possible, before a vaccine becomes available, in order to improve attitudes towards SARS-CoV-2 vaccination. Only by educating the general public about the benefits, safety and efficacy of vaccines can we hope to avoid the unnecessary prolongation of the COVID-19 pandemic. According to the WHO, 26 vaccine candidates are in the process of clinical trials, out of which 6 are already in phase III and 139 are in the process of preclinical evaluation.2 With such a large number of candidates, it is only a matter of time before a safe and effective SARS-CoV-2 vaccine is developed and authorised for worldwide human use. However, after vaccine development, we will be faced with an even more difficult challenge—convincing enough people to actually get vaccinated!Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that has emerged in Wuhan, China in January 2020 and has since spread worldwide and caused a global pandemic with 18 614 177 confirmed cases and 702 642 deaths accordion to the WHO situation report 199 from August 6, 2020.In August 2020, we conducted a search of the PubMed and Statista databases as well as the internet for survey results on the attitudes of the general population towards SARS-CoV-2 vaccination that were conducted in European countries on a nationally representative sample in 2020.3–22 On average, only 58% (n=14 365/24 970) of participants of the collected surveys across Europe were willing/planning to get a SARS-CoV-2 vaccine once it became available, 16% (n=3998/24 970) were neutral and 26% (n=6607/24 970) were not willing/planning to vaccinate against SARS-CoV-2. Public opinion surveys on SARS-CoV-2 vaccination are a valuable tool for approximating the response rate to voluntary vaccination once a vaccine becomes available. We pooled available survey results regarding the public opinion on SARS-CoV-2 vaccination in 26 European countries conducted on a total of 24 970 participants, As can be seen on 0 estimated at 3) and could potentially be reached through the natural course of the disease; however, the expected number of deaths to reach it would exceed 30 million people globally  of 0.6%).23 Serological studies in Spain demonstrated that the seropositivity in the general population was approximately 5%,24 implicating that Spain, a country which was a SARS-CoV-2 hotspot, is still far from reaching the satisfactory herd immunity levels through natural course of the disease and highlighting the importance of urgent vaccine development. The minimum percentage of the population which would have to be vaccinated in order to reach the herd immunity threshold depends mostly on the efficacy of the developed vaccine and will certainly have to be higher than the estimated 67% simply because no vaccine is 100% efficient (most childhood vaccines are effective in 85–95% of recipients.25)Due to the sheer number of potential SARS-CoV-2 vaccines in development, it is safe to presume that not one but many vaccines will be made available and approved in the near future, but even after safe and effective vaccines are developed, the war on SARS-CoV-2 will still be far from over. A plethora of practical and political problems like vaccine distribution and price formation will still remain to be resolved; however, it will all be futile if the general population refuses to participate in the widespread vaccination programmes. The goal of all vaccination programmes is to reach a certain proportion of sufficiently immune population members, without suffering the consequences of natural infection, where the whole population eventually ends up indirectly protected through herd immunity. When a sufficient percentage of population members is immune to the pathogen, the probability that still susceptible individuals encounter an infectious carrier is significantly decreased. The herd immunity threshold for SARS-CoV-2 is approximated to be 67% (R26 leading to a rise in preventable infectious disease outbreaks such as measles.27The collected survey data presented in It is worrisome that on average only 58% (n=14 365/24 970) of the surveyed Europeans were willing to get a SARS-CoV-2 vaccination once it becomes available and that 26% (n=6607/24 970) would refuse to take it altogether. Such public opinion will impede reaching herd immunity levels through vaccination, however, since approximately 16% (n=3998/24 970) of the surveyed remained undecided, a widespread public intervention aimed at increasing the awareness of importance, safety and efficacy of vaccines in control of this and many other infectious diseases could be worthwhile.Although some may argue that Russia is not a European country, we have decided to include it in our survey pool on the basis that the European part of Russia occupies 39% of Europe’s total land area and although this portion comprises only 25% of Russia’s whole land territory, it houses 78% of the Russian population.28 In contrast, a recent United States study reported a 67% acceptance rate for a COVID-19 vaccine, however with noticeable demographic and geographical disparities in vaccine acceptance.29It is also important to understand the reasons why people do not want to get vaccinated. Among the participants of the survey that was conducted in Russia who stated that they would not get a vaccine, 19% stated that they feel no need for it, 18% were concerned about the effectiveness of such a vaccine, 11% were not clearly informed about the new vaccine, 9% regarded the process of vaccination as dangerous, 9% stated the vaccines’ impact on the immune system as a reason not to get vaccinated, 8% did not believe in or wanted to get a vaccine without additional information, 5% did not believe in the existence of SARS-CoV-2, 5% were afraid of vaccination consequences, 4% stated contraindications for vaccination, 4% did not believe that SARS-CoV-2 is dangerous to them and 3% believed they had a good immune system.All the stated reasons emphasise the lack of proper information and trust towards science and vaccination among the members of the general public. Thus, scientists, physicians, public health and government authorities should all strive to address the questions and doubts of the general public and begin providing answers and reassurance in a clear, repetitive and organised way.Global politics play an important role in the development of a SARS-CoV-2 vaccine and one could say that it is the equivalent of the 20th-century space race with governments working as quick as possible to be the first to develop a vaccine, motivated only by the victory in the vaccination race, and not necessarily by the prospect of saving lives. In our view, the global scientific community must not be subjected to political influence and it should remain transparent, honest and selfless. In a race for a vaccine, it is not unimaginable that some may sidestep important safety trials thus potentially endangering their citizens. One potential example of such a rushed vaccination approval is the Russian Sputnik vaccine, which was approved in Russia without a proper phase III trial. Such hurried and politicised vaccines have the potential to further undermine the trust in science and vaccine development and eventually do more harm than good.One of the main limitations of this study is the fact that the public opinion studies for each evaluated country were conducted at different time points in 2020. The extremely small positive response rate in Hungary could be significantly influenced by the fact that the data were collected in February 2020, before the first Hungarian COVID-19 case was confirmed on March 4. The collected survey results represent the public’s opinion only in a certain period of time in which they were performed and can have a limitation of selection bias. The global situation regarding SARS-CoV-2 is not static, thus, the ever-increasing numbers of confirmed cases and fatalities surely have an impact on the public opinion towards SARS-CoV-2 vaccination.30, which provide excellent and cheap means for widespread misinformation distribution; thus, it seems reasonable to also rely on mass media and online social platforms to distribute the positive messages regarding the vaccination benefits. It would also be key to review why the already performed educational campaigns on this topic seem to have failed so far.The exact plans of the suggested public health interventions will have to differ from country to country. The unfortunate global success of the anti-vaccination movement relies heavily on social mediaLike often in science, our study results raise more questions and potential problems than answers. As can be seen, the percentage of people willing to vaccinate against SARS-CoV-2 varies significantly from country to country, ranging from as little as 30% to 80%. The question that remains is whether neighbouring countries with different percentages of vaccinated people will still insist on a negative PCR test or proof of vaccination status for free travel in the future? Will there be significant differences among the developed vaccines  which could influence the general response to vaccination? If the vaccination response remains too low, will we have to quickly change plans and start investing financial and scientific resources more into research of the treatments for COVID-19 or should we have done so from the start in parallel to vaccine development? The list goes on and on, but the only thing that is certain for now is that reaching adequate SARS-CoV-2 vaccination levels globally will be one of the most difficult challenges the health community has faced so far and that the appropriate time to act is now. Only by educating the public about the benefits, safety and efficacy of vaccines can we hope to avoid the prolongation of the COVID-19 pandemic even after vaccines become widely available.Our pooled survey results reveal that on average only 58% of responders across Europe were willing to get a SARS-CoV-2 vaccine once it becomes available.Low vaccination response could make reaching herd immunity to SARS-CoV-2 difficult and unnecessarily prolong the pandemic.We propose the start of educational public health activities on the topic of vaccination as soon as possible, before the vaccine becomes available, in order to improve attitudes towards SARS-CoV-2 vaccination.Why is vaccination hesitancy to SARS-CoV-2 so high in Europe?What public health actions would be most effective in influencing public opinion regarding vaccination?Has the general public’s attitude towards SARS-CoV-2 vaccination changed since this study was conducted?In recent years, vaccine hesitancy has been on the rise, with childhood vaccination rates declining and preventable diseases outbreaks occurring more frequently.Once one or more SARS-CoV-2 vaccines become available, reaching adequately high vaccination levels in the population will be critical for achieving herd immunity.Low vaccination rates against SARS-CoV-2 could have serious medical, social and economic consequences by extending the duration of the pandemic.


Glenoid bone loss remains a challenge in shoulder arthroplasty. Addressing substantial bone loss is essential to ensure proper function and stability of the shoulder prosthesis and to prevent baseplate loosening and subsequent revision surgery. Current options for creating and shaping glenoid bone grafts include free-hand techniques and simple reusable cutting guides that cut the graft at a standard angle. There is currently no patient-specific device available that enables surgeons to accurately prepare the bone graft and correct glenoid deformity. We present a novel surgical technique using three-dimensional (3D)-printed cutting guides to create a patient-specific bone graft to address glenoid deformity in the setting of reverse shoulder arthroplasty. They demonstrated that the use of an angled bone graft resulted in a reliable and predictable union of the graft with the native glenoid, correction of malalignment, and successful lateralization of the glenoid component. This technique has demonstrated good results in the literature with high rates of graft incorporation and decreased complications.12 Such procedures require minimal access to specialized equipment but are technically challenging and time-consuming and fail to address the variability in graft size/shape that is required to address glenoid deformities between patients.11 While there are simple reusable cutting guides available to prepare either a symmetrical or angled bone graft, there is currently no patient-specific device available that enables surgeons to accurately prepare the bone graft and correct glenoid deformity.Boileau et al16 PSI involves creating surgical instruments based on a patient's unique anatomy and using this technology to implant components in an optimal position. This is particularly relevant in cases with severe glenoid deformity or deficient bone stock which increases the risk for suboptimal glenoid component positioning and fixation. SDS and 3D-printed cutting guides are commonly used in other surgical areas that require bone graft reconstruction such as mandibular reconstruction and bone tumor surgery.14 Current options for creating and shaping glenoid bone grafts are limited and include free-hand cutting  or the use of a reusable cutting guide that cuts the graft at a standard size and angle.The use of patient-specific instrumentation (PSI), surgical design and simulation (SDS) and three-dimensional (3D) printing has rapidly evolved in medicine and its use is expanding in certain areas of orthopedic surgery, including but not limited to, orthopedic oncology, arthroplasty, and deformity correction surgery.At our institution, when confronted with substantial glenoid deformity, surgeons have also used SDS and a 3D-print of the patient's scapula and humerus to create a custom  single-use 3D-printed cutting guide. This permits efficient and accurate preparation of the bone graft, utilizing humeral head autograft, to the precise dimensions required to correct each patient's glenoid deformity . The purpose of this study was to present a novel surgical technique utilizing a patient-specific 3D-printed cutting guide to create a customized bone graft to precisely match the glenoid deformity in the setting of RSA with glenoid bone loss.The patient is assessed in the clinic setting and a standard history is obtained and a physical examination is performed taking note of shoulder strength, range of motion, and the presence of lag signs. Plain radiographs consisting of true anteroposterior and axillary views of the shoulder are obtained. Preoperatively, informed consent was obtained from each patient for the publication of the case details and accompanying imaging.21  and angulation. This information is used to obtain a PSI guide to replicate the same implant positioning intraoperatively.A high-resolution computed tomographic (CT) scan of the operative shoulder is obtained to assess glenoid version and inclination . Images are exported in Digital Imaging and Communications in Medicine (DICOM) format to facilitate planning and 3D-printing of the cutting guides. Preoperative planning is performed with 3D planning software  which permits accurate and reproducible measurements of version/inclination and the graft dimensions required to address the glenoid deformity21 . The virOnce the dimensions of the patient-specific bone graft were defined as part of the preoperative plan, single-use patient-specific 3D-printed cutting guides are constructed at the Institute for Reconstructive Sciences in Medicine , an internationally recognized center focusing on reconstructive medicine and the application of advanced digital technology in surgery. Two patient-specific cutting guides are developed in order to create sequential bone cuts of the proximal humerus. Additionally, the patient's scapula and humerus and a replica of the bone graft with the same dimensions as the preoperative plan are 3D printed . The 2 gThe patient's DICOM data are imported into Mimics Medical Version 21.0.0.406 software  to create a 3D model of the patient's scapula and humerus. The graft dimensions are taken from the BLUEPRINT plan and used to create a 3D model using Rhinoceros 3D Version 7 SR16 software (Robert McNeel & Associates). A cylinder is created using the planned diameter of the graft. The minimum and maximum heights and angle are used to determine the cutting plane to create the graft. The cylinder and graft model are taken into Geomagic Freeform Version V2021.1.25 software  and positioned within the 3D model of the humeral head. The position of the graft model within the humeral head can be manipulated as necessary to avoid any areas with poor bone quality or cystic changes seen on preoperative CT. A cutting plane is positioned to create the cut that will provide the depth position and to create a flat surface A and B. After regional  and general anesthesia, the patient is placed in the beach chair position with the head of bed at 30° to 40° of elevation. A standard deltopectoral approach is utilized. The biceps tendon, if present, is either tenotomized or tenodesed to the pectoralis major tendon. A subscapularis peel is performed, which includes careful capsular release along the humeral neck, to expose the glenohumeral joint. With gentle adduction, extension and external rotation of the shoulder, the shoulder is dislocated and the humeral head and proximal humerus exposure is optimized. Next, a series of stepwise bone cuts is performed utilizing 2 3D-printed cutting guides. Minimal osteophyte removal is performed to maintain the native anatomy of the proximal humerus. The first 3D-printed cutting guide is made to fit the exact contour of the proximal humerus, which is secured using 3 smooth 3 mm pins  . An alig22 Care is taken during reaming to preserve the native glenoid deformity to allow proper seating of the harvested graft and thereby optimize baseplate stability. The native glenoid surface that does not undergo reaming is drilled with a 2 mm drill bit to create a bleeding bone surface, which has been demonstrated to aid in bone graft incorporation.23 The patient-specific bone graft is then placed over the central post of the glenoid baseplate, with the deepest portion of the graft properly oriented to match the previously marked “point of maximal defect,” and is then impacted until fully seated . The glenoid surface is then thoroughly assessed and the point of the “maximal glenoid defect” is marked with electrocautery and compared to the 3D-printed glenoid and preoperative plan prior to high-side reaming.y seated  A and B.24 showing 7° of retroversion. Inclination was found to be 2° of inferior inclination using the beta angle method.25 At his 6-week follow-up, the patient was doing excellent with minimal pain. On examination, he was able to achieve 140° of forward flexion and approximately 5° of external rotation.A 69-year-old, right-hand dominant electrician presented with left shoulder pain exacerbated by repetitive work and weakness resulting from end-stage osteoarthritis. He received a contralateral right RSA with bone autograft utilizing a free-hand technique 1 year prior due to ongoing pain and dysfunction. On examination of the left shoulder, there was 90° of active forward flexion, neutral external rotation and internal rotation to the lateral hip. Preoperative planning using 3D CT software revealed 33° of glenoid retroversion and 3° of superior inclination. Our goal for correction determined by the PSI guide was 7° of retroversion and 3° of inferior inclination. The dimensions of the bone graft to achieve this correction were calculated to be 29 mm in diameter, 12 mm in height, with an angle of 28.4°. While performing the preoperative design of the cutting guide, multiple cysts in the humeral head were noted. The initial planned resection planes would have resulted in a wedge of bone through this area of poor-quality bone. Utilizing the planning software, the planned wedge of bone was rotated and translated medially in order to avoid this cystic area. The patient underwent an RSA with bone grafting utilizing the above technique with 3D-printed cutting guides without complication. Preoperative and postoperative radiographs and CT are demonstrated in 9 Substantial glenoid bone loss in the setting of primary or revision arthroplasty has been associated with inferior outcomes,7 raising several potential issues including component instability, mechanical impingement and scapular notching, poor soft-tissue tensioning and limited options for revision procedures. Glenoid defect patterns are determined based on their etiology. Glenohumeral arthritis is associated with posterior wear,26 rotator cuff arthropathy with posterosuperior wear,27 chronic anterior instability with anterior wear28 and revision cases with universal bone loss.29 Walch et al26 described the most commonly used classification system for glenoid erosion, as follows: A1, concentric; A2, concentric and centrally eroded; B1, posteriorly subluxated; B2, posteriorly subluxated and eroded; and C, retroverted and hypoplastic. Sirveaux et al30 expanded upon this and created a classification system to describe superior glenoid wear in patients with CTA: Grade E0, no wear; E1, central concentric wear; E2 and E3, superior wear; and E4, inferior glenoid erosion.Acquired glenoid bone loss is found in up to 39% of patients undergoing an RSA with 15% requiring bone grafting.33 At our institution, utilizing an angled bone graft harvested from the humeral head  is the preferred technique as it offers a biologic solution with the advantages of addressing the glenoid defect, reconstituting the joint line and increasing the available bone stock for future revision scenarios.34 A recent systematic review by Malahias et al35 including 13 studies (539 shoulders) on glenoid bone grafting in RSA concluded that bone grafting the glenoid with autograft or allograft bone is associated with satisfactory clinical function and radiographic outcomes in the short-term with a low rate of complications requiring surgery (3.5%). In the primary setting, rate of radiographic graft nonunion was 5.2%, glenoid component migration was 0.4% and infection rate was 0.9%. Additionally, Lanham et al36 published a systematic review including 19 studies (652 shoulders) comparing bone grafting and augmented glenoid baseplates and found that both groups had similar clinical outcomes, complications and revision rates; however, there was a slightly higher rate of infection and scapular notching in the bone grafting group. An increased rate of infection when performing bone grafting has been postulated to be due to the increased surgical time that is required to create the bone graft, as increased surgical time has been shown to increase the rate of prosthetic joint infections in hip and knee arthroplasty.37 Although utilizing patient-specific cutting guides increases the time needed for preoperative planning, we hypothesize the intraoperative time required to create such grafts will be decreased. More data is required, however, to test this hypothesis.In order to address glenoid erosion, multiple techniques have been described including eccentric reaming, trabecular metal augmented glenoid baseplates, and bone grafting.40 Our goal is to implant the baseplate within 10° of neutral version and full correction of glenoid inclination . Using these parameters during the preoperative planning stage, we can determine the exact dimensions of a graft required for each patient . Multiple studies have assessed the accuracy of glenoid baseplate implantation utilizing 3D planning software. Iannotti et al41 determined that the use of standardized instrumentation and 3D planning improved guide pin positioning compared to standard instruments and two-dimensional planning in anatomic shoulder arthroplasty. This finding was replicated in patients requiring RSA and bone grafting.42 In contrast, whether glenoid PSI guides are necessary to improve the positioning of the glenoid baseplate in cases with glenoid deformity has more conflicting results. Eraly et al43 created glenoid defects in 10 cadaveric shoulders and implanted an RSA using patient-specific guides versus standard instrumentation. The patient-specific guides revealed significant reductions in angular deviation from the planned glenoid implant position. In addition, the use of the intraoperative guide permitted surgeons to obtain 89% of the preoperatively planned intraosseous screw length compared with 52% in the group without a guide. Tashjian42 compared baseplate positioning in 15 patients requiring a bone graft using 3D planning and standard instrumentation versus 3D planning and a PSI guide. Although a significant difference in final retroversion of 6° was found, this value was not deemed clinically meaningful and recommended that PSI guides may be more useful to address severe deformities . Although there may be conflicting results in regard to the necessity of PSI guides in shoulder arthroplasty, our experience has been favorable and therefore we recommend their use as an integral step in this surgical technique to ensure proper base plate positioning and subsequently correct positioning of the bone graft on the prepared glenoid in order to restore glenoid alignment.Preoperative planning with 3D planning software is a crucial step in this technique to ensure an accurate assessment of the glenoid bone loss and bone graft required to address the deformity and positioning of the glenoid baseplate.47 Procedures that utilize these guides such as in mandibular reconstructive surgery have demonstrated reduced surgical time and overall improvements in the accuracy, safety, and reliability of these procedures.15 In the field of orthopedic surgery, patient-specific cutting guides have been used for the distal femur, proximal tibia,13 pelvis14 and the midfoot,15 and have demonstrated excellent surgical accuracy, ease of use, and superior results over free-hand techniques. Additionally, a study by Jaffry et al16 demonstrated that the use of 3D-printed instruments for unicompartmental knee arthroplasty revealed a good correlation with implants placed by robot-assisted surgery.The medical applications of 3D printing are rapidly evolving. 3D-printed guides have been extensively used in maxillary and particularly mandibular reconstructive surgery to replace what was previously considered a gold standard in treatment using fibular osseous vascular free flaps.To our knowledge, the use of 3D-printed cutting guides has not been described in shoulder arthroplasty. Although the outcomes have not yet been delineated, we see many potential advantages to this technique. The cutting guides allow for precise replication of the bone graft that was templated preoperatively using 3D planning software to ensure accurate correction of the glenoid deformity and appropriate glenoid baseplate positioning. Preoperatively, we are able to modify which area the cutting guide harvests the wedge of bone from on the humeral head in order to avoid areas of cystic changes. Furthermore, by presenting a standardized technique to create a bone graft, we anticipate that the overall surgical time for this step of the procedure will be decreased, which in turn, may decrease postoperative infection rates. However, short- and long-term studies are required to verify these potential advantages.Potential limitations of this novel technique include the necessary access to an institution that has the ability to 3D print and manufacture the cutting guides required to create the bone graft, thereby limiting the generalizability of this technique to other surgical centers. The preoperative planning phase of this technique takes longer than when using standard instrumentation due to the time required to obtain advanced imaging and a PSI guide, and the time needed to create the 3D-printed guides. Additionally, a specific limitation in our healthcare system is the time lag between acquiring the initial preoperative CT and the surgical date. If this time is lengthy , the patient's anatomy may subtly change with the progression of arthritis, which may compromise the exact fit of the cutting guides on the proximal humerus in addition to the dimensions of the bone graft that is required to address the glenoid deformity. Finally, it is yet to be determined whether this technique will be accurate and clinically relevant in the short- and long-term compared to standard instrumentation. In the case example presented here, only short-term results are available; long-term clinical and radiographic outcome data is required to determine the efficacy of this technique.The authors have described a novel technique using 3D-printed cutting guides to create a patient-specific bone graft to address severe glenoid deformity in the setting of RSA.


The results revealed that GNAI3 is associated with a poor prognosis and is involved in significantly important pathways, such as macrophage maturation and cytoskeleton arrangements. These findings suggest that GNAI3 may serve as a valuable prognostic biomarker for the GBM microenvironment and could provide actionable information for the treatment of GBM.In this study, we aimed to address the critical need for a prognostic biomarker in the treatment of GBM. Various approaches and treatments have been examined in the recent literature; however, their effectiveness is limited due to the highly invasive, heterogeneous, and resistant nature of GBM tumors. TCGA, which is a database available online, was used to assess the role of guanine nucleotide-binding protein G(i) subunit alpha 3 GNAI) family of genes in GBM using a Chinese Glioma Genome Atlas (CGGA) dataset. Within this dataset, we observed the association in the tumor microenvironment between the gene expression of GNAI subunit 3 (GNAI3) and a poor prognosis. MetaCore and gene ontology (GO) analyses were conducted to explore the role of GNAI3 in co-expressed genes and associated signaling pathways using a transcript analysis. Notable pathways included “Cytoskeleton remodeling regulation of actin cytoskeleton organization by the kinase effectors of Rho GTPases” and “Immune response B cell antigen receptor (BCR) pathway”. A single-cell analysis was used to assess GNAI3 expression in GBM. The results demonstrated that GNAI family genes, specifically GNAI3, were significantly associated with carcinogenesis and malignancy in GBM patients. Our findings suggest that the GNAI3 gene holds potential as a prognostic biomarker for GBM.Glioblastoma multiforme (GBM) is one of the most aggressive cancers with a low overall survival rate. The treatment of GBM is challenging due to the presence of the blood–brain barrier (BBB), which hinders drug delivery. Invasive procedures alone are not effective at completely removing such tumors. Hence, identifying the crucial pathways and biomarkers for the treatment of GBM is of prime importance. We conducted this study to identify the pathways associated with GBM. We used The Cancer Genome Atlas (TCGA) GBM genomic dataset to identify differentially expressed genes (DEGs). We investigated the prognostic values of the guanine nucleotide-binding protein G(i) alpha subunit ( Glioblastoma, commonly known as glioblastoma multiforme (GBM), is an exceedingly aggressive and lethal type of brain tumor that originates from glial cells, which are supportive cells in the brain . GBM usuGNAI family, with a particular focus on the guanine nucleotide-binding protein G(i) subunit alpha 3 (GNAI3), represents a noteworthy protein family of interest . We. WeGNAI GNAI family genes interacted with each other, and the other members of the guanine nucleotide-binding (GNB) family were involved in cell cycle responses and signaling pathways , except for austocystin D. TKIs target the epidermal growth factor receptor (EGFR) and Erb-B2 Receptor Tyrosine Kinase 2 (ERRB2) pathways, which are known to play crucial roles in tumor progression and metastasis. Austocystin D functions as an inducer of DNA damage and targets nuclear receptor subfamily 1 . The GDSGNAI2 and GNAI3 were examined for GBM using immunohistochemical (IHC) scores from HPA. The IHC images included clinico-pathological information, such as the patient’s age, gender, and ID, as well as normal and tumor samples. In the analysis, the GNAI2 protein was not significantly or differentially expressed between the normal and tumor tissues. The expression of the GNAI3 protein was higher in the tumor tissues. The protein expression levels were quantified using a score that was automatically determined based on a combination of the staining fraction and intensity. The scoring system included categories such as negative (not detected), weak (<25% (low)), moderate (<25% (medium)), and strong (<25% (medium) to <25% (high)), with corresponding definitions for combined fractions and intensities.After performing the survival analysis, the protein expressions of The analysis of the HPA results revealed a strong association between GNAI3 and GBM in the staining samples. The expression of GNAI2 was less distinct, suggesting that it may have a limited role as a prognostic biomarker for GBM. GNAI3 in brain cancer cell lines was observed to be notably higher and more significant in the DOAY, U87MG ATCC, and SF172 cell lines than in the other cell lines (n = 65), as shown in The expression of GNAI3 for further analysis. GNAI3 demonstrated statistical significance in primary and recurrent gliomas; consequently, we conducted an in-depth analysis to assess its role as a potential prognostic biomarker for GBM because it demonstrated a high mRNA expression that was highly significant. It also exhibited an increased expression as the tumor grew; a higher expression of this gene led to poor survival outcomes due to the poor prognostic value.Based on the IHC staining and differential expression between normal and cancer tissues, we focused on GNAI3 demonstrated an involvement in nuclear speckles and spliceosomal complexes in the CCs. The BP pathways included ribonucleoprotein complex biogenesis, ribosome biogenesis, non-coding (nc) RNA processing, and RNA splicing. The associated KEGG pathways included spliceosomes, nucleo-cytoplasmic transport, and ribosome biogenesis in eukaryotes  analysis was conducted using the R software 4.3.1 clusterprofiler package for a pathway analysis. Spearman’s correlation (>0.45) was used as a threshold; 127 genes were input for the GO analyses using a co-expression dataset obtained from cBioPortal. GNAI3 was associated with catalytic activity, acting on RNA and ribonucleoprotein complex binding in the MFs. karyotes . These rGNAI3 in GBM. The tumor microenvironment (TME) is a changing and emergent entity; it varies among cancer types. Research on the TME is ongoing, but existing research has demonstrated the importance of the TME in cancer growth and progression [GNAI3. It demonstrated a positive correlation with myeloid DCs . It was negatively correlated with B cells , CD4+ T cells , macrophages , neutrophils , and CD8+ T cells  (We used the TIMER database to determine immune infiltration associated with gression . To unde × 10−1) . The rolGNAI3 using the co-expressed gene dataset from TCGA. It revealed that GNAI3 was involved in various significant pathways that regulate BPs, such as “cytoskeleton remodeling regulation of actin cytoskeleton organization by kinase effectors of Rho GTPases”  is a protein that produces contractile filaments of muscle cells and is involved in several cellular movements, including cell motility, cell structure and integrity, and intracellular trafficking. Cellular transformation, including the genesis and growth of cancer cells, is typically accompanied by changes in the cytoskeleton and organization of the actin structure. GNAI3 may play a role in the onset or progression of GBM by altering cellular structures and behaviors [GNAI3 expression is implicated in pathway changes, with GBM progression from lower (less severe) to higher (more severe) WHO scores. This may reveal how GNAI3, as a biomarker, necessitates the consideration of its expression levels in GBM compared with healthy tissues.In terms of the involvement of ehaviors . SignifiGNAI3 and DCs in the setting of infiltrating the immune cells of GBM may imply that enhanced GNAI3 signaling promotes DC recruitment or activity. DCs are antigen-presenting cells (APCs) that play a key role in initiating the adaptive immune response. They may boost antitumor responses by delivering tumor antigens and activating T cells. Negative correlations of GNAI3 with B cells, CD4+ T cells, CD8+ T cells, macrophages, and neutrophils imply that GNAI3 signaling may impede the recruitment or function of those cells [GNAI3 is also involved in signal transduction. It is implicated in several intracellular signaling pathways; the B-cell antigen receptor (BCR) pathway is critical for the activation and function of B cells. When an antigen binds to the BCR, it initiates a series of intracellular signaling events that result in the activation, proliferation, differentiation, and antibody production of B cells. Notably, while the BCR and GPCR pathways are distinct, but they may overlap. Several studies have revealed that G proteins play a role in regulating BCR signaling, altering B-cell activation and function. Little is known about the involvement of GNAI3 with B-cell function in the context of GBM. B cells are part of a complex immune response to GBM, but their specific roles and interactions in the GBM TME remain unknown. Additional research is required to determine how GNAI3 affects B-cell functions and immunological responses in GBM [The positive correlation between se cells ,45. GNAIs in GBM . APCs cos in GBM ,48.GNAI3, can regulate cell proliferation and survival; both are important in carcinogenesis and tumor progression. They also play roles in cell migration and invasion, which are critical for metastasis [GNAI3, are known to play roles in regulating immune cells in the setting of immunological responses. Alterations in this pathway may influence immune responses to GBM. Our study’s limitation is that we did not conduct a thorough verification using in vitro and in vivo experimental assays, but we believe that the findings of the bioinformatics analysis serve as a platform for future research. GNAI3 has the potential to be a biomarker for immune infiltration in the development of GBM malignancies. Data from the single-cell analysis revealed a greater expression in macrophages, indicating that they play an important role in the malignancy of GBM [GNAI3 could be used as a biomarker or a particular target in glioma immunotherapy. Although this is an early study, it is clear that there are significant limitations to how GNAI3 may be altered and regulated with medicines. Further experiments should be performed to investigate the effects of GNAI3 on immune cells within the TME in gliomas to develop personalized approaches such as vaccinations and to explore multiple clinical trials investigating immunotherapy combination studies.G proteins, including tastasis . GPCRs, y of GBM . A high y of GBM . We reveGNAI3 gene could be an indicator of a poor prognosis and a potential biomarker for GBM. A bioinformatics analysis, as well as MetaCore and single-cell analyses, revealed that it is involved in the cellular responses that trigger tumor progression and invasion. This biomarker could be the subject of future in-depth experimental validations. Our results demonstrate that the overexpression of the


The mechanism by which this blunted response to hypoxia occurs is unknown. We have previously shown that a computational model  of the respiratory neural network can be used to test hypotheses focused on changes in chemosensory inputs to the central pattern generator (CPG). We hypothesize that altered chemosensory function at the level of the carotid bodies and/or the nucleus tractus solitarii are responsible for the blunted response to hypoxia. Here, we use our model to explore this hypothesis by altering the properties of the gain function representing oxygen sensing inputs to the CPG. We then vary other parameters in the model and show that oxygen carrying capacity is the most salient factor for producing silent hypoxemia. We call for clinicians to measure hematocrit as a clinical index of altered physiology in response to COVID-19 infection.Silent hypoxemia, or ‘happy hypoxia’, is a puzzling phenomenon in which patients who have contracted COVID-19 exhibit very low oxygen saturation (SaO PaCO2) levels. Although some individuals with COVID-19-induced hypoxemia experience dyspnea (breathing discomfort), many do not  resulted in a progressive increase in respiratory rhythm that showed periodic, multi-periodic, quasi-periodic, and finally chaotic rhythmic patterns [in vivo. Thus, there is precendent for increasing excitability in the respiratory network resulting in a kind of “depolarization blockade” of normal breathing and a cessation of gas exchange that then results in a precipitous fall in PaO2. We described experiments related to this concept in [In addition to the limitations to our model that we have mentioned previously, we realize that our model represents a very reduced number of the elements in the central pattern generator and pattern formation network for breathing control. The brainstem network includes hundreds of neurons that participate in each breath , 67, andpatterns . As excincept in . Because2 and hypercapnea [In conclusion, we call for data to be collected on hematocrit in COVID-19 patients, and testing of metabolism and mitochondrial function. Our model predicts changes in oxygen handling and metabolism in silent hypoxemia patients. In addition, we believe the following measures may have untapped predictive value: minute ventilation, oxygen saturation, and breathing frequency. We speculate that some combination of these quantities, if measured on entry to the ER, could help predict the need for ventilator support in the subsequent 48 hours. We also note that there is a need for incorporating oxygen handling dynamics into more sophisticated state-of-the-art respiratory control models, most of which currently focus on COercapnea . FinallyHere we provide the equations for the 7D-O2 model introduced in .A variety of models have been proposed for the central neural circuits generating breathing rhythms, ranging from group-pacemaker networks to individual pacemaker models, and beyond. Here, we adopt the original Butera-Rinzel-Smith (BRS) model (referred to as “model 1” in ) proposeThe output of the CPG is the BRS cell’s membrane potential d Kp=5mV .The output of the motor unit determines the rise and fall of lung volume le range . We notele range PAO2) increases at a rate given by the pressure difference between external and internal air, and by the lung volume. In contrast, during exhalation 2 between the lungs (PAO2) and the arterial blood PaO2. The rate of change in PaO2 is thus given by:At standard atmospheric pressure (760 mmHg), external air with 21% oxygen content will register a partial pressure of oxygen of PaO2, we write2 per millisecond. The denominator converts changes in the number of moles of O2 in the blood to changes in PaO2. To calculate the flux 2, To represent the change in PaO2. The blood solubility coefficient, 2 at physiological partial pressures 2:Note that the term 2.5 from .2 per gram:Our model includes a parameter PaO2 and drive the central rhythm generator, as described in more detail in [PaO2 as a sigmoidal saturing function, with the firing rate low until PaO2 is reduced below a threshold  and then steep firing rate increases as PaO2 is reduced further [PaO2 with the conductance representing external drive to the CPG Chemosensory feedback from pheripheral chemoreceptors in the carotid bodies, carried to brainstem respiratory circuits via the carotid sinus nerve, close the control loop in our model. These receptors detect reductions in etail in . We mode further , 37. We ode15s in MATLAB).We numerically integrated the preceding equations using a variable-order, variable-step stiff solver (


After cardiac arrest (CA), some patients report impressions with highly realistic features, often referred to as near-death experience (NDE). The frequency of such episodes seems to be variable, with various types of content. In a prospective study, we subjected 126 CA cases treated at the Department of Emergency Medicine of the Medical University of Vienna under carefully controlled conditions to a structured interview. We included all patients admitted due to CA, whose communicative abilities were restored and who agreed to participate in the study. The questionnaire inquired as to living conditions, attitudes towards issues of life and death, and last recollections before and first impressions after the CA. The majority of the subjects (91 = 76%) replied to inquiries concerning impressions during CA with “nothing” or “blackout”, but 20 (16%) gave a detailed account. A German version of the Greyson questionnaire specifically addressing NDE phenomena (included towards the end of the interview) resulted in ≥7 points in five patients (4%). Three patients reported a meeting with deceased relatives (one with 6 Greyson points), one an out-of-body episode, and one having been sucked into a colorful tunnel. Eleven of these twenty cases had their cardiopulmonary resuscitation (CPR) started within the first min of CA, a higher fraction than cases without experience. Reported experience after CA was of high significance for the patients; many of them changed their point of view on issues of life and death. To puterience; ). Duringerience; . On the Our main motivation to embark into this study was the prospect to encounter unusual cognitive phenomena, triggered by the transient cessation of energy supply to the human brain. We expected a low frequency of such events and that, in case they occurred, they could be traced to natural origins. One year and 23 interviews later, we had not seen a single interesting case. During the next 7 years of the project, we were rewarded for our patience with several impressive reports. At the end, we even had reason to examine the presentation history of the hidden image generator.The study started as participation in the multicenter project AWARE . After tPatients were acute admissions to the Department of Emergency Medicine for heart problems. The majority of these problems consisted in CA suffered at home (35.7%), in a hospital (13.5%), on the street (10.3%), or during sport (8.7%). Out-of-hospital CAs were followed by at least one further CA at the emergency unit. Most patients either suffered their CA in their later professional life or a few years after retirement (mean age 58 ± 13 years). The restoration of spontaneous cardiac activity was often followed by physical cooling to 34–32 °C for 12–24 h [At an elevated position above one emergency bed (2 m above ground), a notebook PC was fixed facing the ceiling and displaying images selected at random from a pool of 29, switching from the actual to any in the pool every few hours (the number of hours was unpredictable). These images were not disclosed to the public and were not even known to all of us (in particular not to the main interviewer M.L.B.). The presentation history was stored on the PC, and any readout of this history, be it authorized or not, left its trace. Only one case gave us reason to examine the image in question. Readout was effectuated after closing the study in 2021; no other readout was noticed.We adhered to the questionnaire of the preceding study  with mi. It consPersonal construction . The fraction of patients with recollections (see next paragraph) seemed to increase slightly with the time elapsed between CA and interview . If possA. Kelly ) may havp = 0.199). For more detailed interview summaries than in the table, see A widely used instrument to investigate subjective experience in resuscitated patients is the questionnaire developed by Bruce Greyson in 1983  on the b2 test  the limiting probability p was obtained. For two categories, R.A. Fisher’s exact test was used, several online calculators all returning precisely the same result.To estimate the probability that cases distributed equally into several categories, we subjected the numbers to a χ-squared ). By conp = 0.78) had lost at least 1 day from their memory store. Thus, it is possible to keep in mind perceptions during or shortly after a CA while forgetting others preceding the same CA .A few cases suffered severe losses in memories for events preceding their CA (retrograde amnesia). This occurred to several blackout cases, but also four cases with recollections For more than 5 years, a notebook above one emergency bed displayed image after image to the naked ceiling above without any consequences, but in late August 2017, patient K  was installed on the bed see . It took2 = 13.2, 3 degrees of freedom, p < 0.005).In the first round of questions, patients were asked about religious conviction and attitude towards central features of belief (questions B5–B10). While the clientele of the main Viennese hospital still adhered in large part to Catholicism (39% of our cohort), the fraction declaring no religion came close to this figure (36%). Four cases with detailed recollections  were of Catholic (A and T), Islamic (O), or no conviction (J). Most subjects (91) ascertained they would have given the same answers before their CA, but a minority of 16 underwent a change in mind . Four ca2 6.46, 6 degrees of freedom, p > 0.25). Thirteen ‘experiencers’ were familiar with NDE stereotypes, but four were not, quite close to the situation in the whole sample (65%/22%). An impressive example was case T, a 46-y-old female with a Greyson score of 18 out of 32 possible points, but without ever having heard about NDEs before. Only after her strong experience, she began to search the internet for such reports, with high motivation and interest.This question in the last section of the questionnaire should safeguard against any bias by media influence (question E3). No such bias was found  failed to identify some impressive reports as NDE; only five cases passed as NDE in the strict sense. The faster a patient was subjected to CPR measures, the greater the likelihood of a detailed account. In comparison to other studies , this stSome acute clinical conditions seem to favor subjective experience with qualities indistinguishable from reality. Thonnard et al.  subjecteAs for the paradox of seeing oneself from an apparently extra-corporal point of view (case J), clinical observations at the Geneva University Hospital ,29 suggeOur observation that detailed impressions were more often reported by patients undergoing resuscitation efforts quickly agrees with the idea that they were triggered by oxygen-loaded blood, reaching targets ready to resume cooperation. Technical improvements may allow in the near future the simultaneous recording of local functional activity in the brain during acute CPR efforts by the application of near-infrared spectroscopy and EEG probes, if they are easy to position and robust against artifacts. Actually, the application of such probes still requires too much attention and may divert from urgent life-saving measures.None of us can provide definite proof for the existence of any kind of reality. This epistemological fact was brought forward almost 2.4 millennia ago  for the