More importantly, owing to the positively charged surfaces, both the TFC-PEI-blank and TFN-PEI-GO membranes exhibited excellent rejections toward various heavy metal ions including Zn2+, Cd2+, Cu2+, Ni2+, and Pb2+. Additionally, compared with the negatively charged polypiperazine amide NF membrane, both the TFC-PEI-blank and TFN-PEI-GO-20 membranes demonstrated superior antifouling performance toward the cationic surfactants and basic protein due to their hydrophilic, smooth, and positively charged surface. Moreover, the TFN-PEI-GO membranes presented the improved chlorine resistances with the increasing GO concentration.Using polyethylenimine (PEI) as the aqueous reactive monomers, a positively charged thin-film nanocomposite (TFN) nanofiltration (NF) membrane with enhanced performance was developed by successfully incorporating graphene oxide (GO) into the active layer. The effects of GO concentrations on the surface roughness, water contact angle, water flux, salt rejection, heavy metal removals, antifouling property, and chlorine resistance of the TFN membranes were evaluated in depth. The addition of 20 ppm GO facilitated the formation of thin, smooth, and hydrophilic nanocomposite active layers. Thus, the TFN-PEI-GO-20 membrane showed the optimal water flux of 70.3 L·m Nowadays, many industries such as mining , metalluIn the past few decades, various methods have been applied to remove or reclaim heavy metals, including chemical precipitation , ion excCurrently, most of the commercially available NF membranes are polyamides (PA) thin-film composite (TFC) membranes fabricated through interfacial polymerization (IP) between piperazine (PIP) and trimesoyl chloride (TMC) ,20,21. TPolyethylenimine (PEI) is a known cationic polyelectrolyte with branched chain, containing primary amine groups, secondary amine groups, and tertiary amine groups . PEI hasTo overcome these challenges, novel thin-film nanocomposite (TFN) NF membranes have been extensively studied by incorporating various nanofillers into the active layer ,37,38. IIn this study, in order to construct the positively charged NF membrane with enhanced performance, PEI and GO were used as the aqueous reactive monomers and the aqueous co-additives, respectively. The effects of GO concentrations on the resultant positively charged NF membranes were investigated in depth in terms of the surface roughness, water contact angle, water flux, salt rejection, heavy metal removals, antifouling property, and chlorine resistance. This novel, positively charged NF membrane demonstrated an exceptional removal of heavy metal ions with improved antifouling performance and chlorine resistance.Mw = 2000), and 4-dimethylaminopyridine  were purchased from Shanghai Macklin Biochemical Company, Shanghai, China. Triethylamine , n-hexane, ethanol, sulfuric acid , hydrochloric acid , hydrogen peroxide , sodium nitrate , sodium hypochlorite , sodium sulfate , magnesium sulfate , sodium chloride , magnesium chloride , copper chloride (CuCl2), nickel chloride (NiCl2), zinc chloride (ZnCl2), cadmium chloride (CdCl2), and lead nitrate (Pb(NO3)2) were purchased from Sinopharm Chemical Reagent Company, Shanghai, China. Lysozyme was obtained from Dingguo Changsheng Company, Beijing, China.Analytical grade piperazine , trimesoyl chloride , cetyl trimethyl ammonium chloride , dodecyl trimethylammonium chloride , PEI  UF membrane was lab-made and used as the supporting layer in our previous report . The PSUThe cross-sectional and surface morphologies of the fabricated membranes were observed by SEM . The surface roughness was recorded by atomic force microscopy . The chemical groups of the membrane surfaces were characterized by attenuated total reflectance-Fourier transform infrared spectroscopy . The chemical compositions of the membrane surfaces were characterized by XPS . The membrane hydrophilicity was evaluated by a contact angle goniometer .2 effective area . The tested membranes were prepressured for 30 min under the transmembrane pressure of 0.5 MPa, the temperature of 25 °C, and the tangential velocity of 1.8 m/s. The salt rejections were first evaluated using four kinds of 2000 ppm salt solutions . The removals of heavy metal ions were further evaluated using five kinds of 500 ppm heavy metal salt solutions 2). The salt rejections were measured under the full batch mode (both the concentration and permeate stream were recycled into the feed tank) to keep the feed concentration constant. The key parameters of the related ions are listed in The membrane separation performance was tested using a laboratory cross-flow membrane separation device with 70 cmJ is the water flux , V is the volume of the permeate (L), A is the effective area of the membrane (m2), and Δt is the filtration time (h).pC and cC refer to the solute concentration in the permeate and in the concentrate, respectively. The salt concentration was calculated according to the standard curve between the concentration and the conductivity measured by a conductivity meter .The water flux and rejection were calculated by Equations (1) and (2), respectively:Two kinds of cationic surfactants  and the basic protein (lysozyme) were used as the model foulants to evaluate the antifouling performance of the selected NF membranes. First, the pure water flux was recorded for 60 min at 0.5 MPa. Then, the pure water was replaced with a 2000 ppm solution containing the model foulant, and the flux of the pollutant solution was recorded for an additional 60 min. After water washing, both the water filtration procedure and pollutants’ filtration procedure were repeated.After immersion in a 2000-ppm NaClO solution, the selected NF membranes were remeasured in terms of water flux and salt rejection. The chlorine resistances were evaluated based on the flux variation and the rejection variation.The surface and cross-section morphologies of the fabricated NF membranes were characterized by SEM, as shown in According to the cross-sectional SEM images (Ra), the root mean square of Z data (Rq), and the mean difference between the five highest peaks and five lowest valleys (Rz). According to The surface roughness of the fabricated NF membranes was further characterized by AFM. The key parameters of surface roughness include the mean roughness  is commonly used to evaluate membrane hydrophilicity. The relationship between the GO concentration and the WCA of the composite NF membranes is depicted in 2SO4 rejections, above 91%, regardless of the variation of GO concentration. This suggested that the introduction of GO improved water flux without sacrificing the salt rejection. The increase of water flux benefitted from: (1) The introduction of GO generated a thinner active layer, which correspondingly alleviated the membrane filtration resistance; (2) the introduction of GO led to the formation of the wrinkled membrane surface, which increased the effective membrane filtration area; (3) the hydrophilicity of the active layer was enhanced with embedding GO; and (4) GO provided additional transmission channels with less mass transfer resistance to water molecules.The water fluxes and salt rejections of composite NF membranes were measured and are shown in 2 ≈ MgSO4 > Na2SO4 > NaCl, which matched very well with the characteristics of the positively charged NF membrane [In order to explore in depth the separation mechanisms of the positively charged PEI-based NF membranes, the rejections of four kinds of salts were measured and are illustrated in membrane . This or2+ than the monovalent Na+. As a result, the rejection of divalent Mg2+ was greater than that of monovalent Na+. For the salts having the same divalent Mg2+, the positively charged TFN-PEI-GO membranes also produced the stronger electrostatic attraction toward the divalent SO42− than the monovalent Cl−. The strong adsorption of SO42− significantly weakened the positively charged properties of membrane surfaces. As a result, the rejection of MgSO4 was lower than that of MgCl2. However, for the salts having the same monovalent Na+, the Na2SO4 rejection was much higher than NaCl rejection because SO42− has a greater hydration radius than Cl−, which indicated that the size exclusion effect was more dominant than the Donnan exclusion effect.It is well known that the NF separation mechanisms include the Donnan exclusion effect and the size exclusion effect. Therefore, the positively charged TFN-PEI-GO membranes generated the stronger electrostatic repulsion toward the divalent Mg3)2 was 91% and the rejections of the other heavy metal salts ranged from 94% to 97%. This indicated that the positively charged NF membranes fabricated from PEI monomers were helpful to enhance the removals of heavy metals. Because both the TFC-PEI-blank membrane and the TFN-PEI-GO membranes were positively charged, these membranes presented the same sequence for removing heavy metals: ZnCl2 > CdCl2 ≈ CuCl2 > NiCl2 > Pb(NO3)2. This result was consistent with the hydration radius sequence of various heavy metal ions, as listed in 2+ > Cd2+ > Cu2+ > Ni2+ > Pb2+. Five kinds of heavy metal ions belong to divalent cations. Thus, the rejections depended on the hydration radius based on size exclusion effect. The larger hydration radius of heavy metal ion contributed to the high rejection of heavy metal ion.The TFC-PEI-blank, TFN-PEI-GO-20, and TFN-PEI-GO-40 were selected to investigate their separation performance of removing heavy metals. According to 4 or CuCl2, the Cu2+ rejection of the positively charged TFC-PEI-blank membrane was remarkably higher than that of the negatively charged TFC-PIP-blank membrane. For the TFC-PEI-blank membrane, the rejections of both CuSO4 and CuCl2 were above 94%, and the CuSO4 rejection was slightly lower than the CuCl2 rejection because the positively charged TFC-PEI-blank membrane generated the stronger electrostatic attraction toward the divalent SO42− than the monovalent Cl−. This suggested that the Donnan exclusion effect was predominant compared with the size exclusion effect. On the contrary, the negatively charged TFC-PIP-blank membrane presented the stronger electrostatic repulsion toward the divalent SO42− than the monovalent Cl−. Thus, both the Donnan exclusion and size exclusion preferred to increase the CuSO4 rejection rather than the CuCl2 rejection for the TFC-PIP-blank membrane.The influences of different aqueous reactive monomers (PIP vs. PEI) on the copper removal of the TFC NF membranes were further investigated. As shown in Two kinds of cationic surfactant (CTAC and DTAC) and lysozyme (LYZ) were used to evaluate the antifouling performance of the fabricated NF membranes. According to After fouling by CTAC and water washing, the normalized water fluxes of the TFC-PEI-blank and TFN-PEI-GO-20 membranes reached 91.1% and 89.7%, respectively. However, the normalized water flux of the TFC-PIP-blank membrane was only 76%. After fouling by DTAC, the normalized water fluxes of the TFC-PEI-blank and TFN-PEI-GO-20 membranes reached 85.9% and 85.3%, respectively. However, the normalized water flux of TFC-PIP-blank was only 75.4%. LYZ is positively charged under neutral testing conditions due to its isoelectric point of 10.7. After fouling by LYZ, the normalized water fluxes of the TFC-PEI-blank and TFN-PEI-GO-20 membranes reached 85.0% and 83.1%, respectively, while the normalized water flux of the TFC-PIP-blank membrane was only 62.8%. Therefore, for the positively charged foulants, the above results indicated that the positively charged TFC-PEI-blank and TFN-PEI-GO-20 membranes had superior antifouling performance over the negatively charged TFC-PIP-blank membrane.On the one hand, both the TFC-PEI-blank and TFN-PEI-GO-20 membranes possessed a more hydrophilic and smoother surface than the TFC-PIP-blank membrane, which was helpful to reduce membrane fouling. On the other hand, the positively charged TFC-PEI-blank and TFN-PEI-GO-20 membranes generated the stronger electrostatic repulsion toward the cationic surfactants and the positively charged LYZ, which further alleviated the membrane fouling. On the contrary, the negatively charged TFC-PIP-blank membrane had the strong electrostatic attraction toward the cationic surfactants and the positively charged LYZ, which resulted in the severe membrane fouling.Although the TFN-PEI-GO-20 membrane was more hydrophilic compared with the TFC-PEI-blank membrane, the TFN-PEI-GO-20 membrane presented a slightly more severe membrane fouling than the TFC-PEI-blank membrane. This was caused by the higher flux of the TFN-PEI-GO-20 membrane than that of the TFC-PEI-blank membrane. It means that more foulants were adsorbed on the membrane surface and the concentration polarization was intensified due to the high membrane flux, which would aggravate membrane fouling.In view of the degradation mechanism of the PA TFC membranes, the PEI-based PA membranes are vulnerable to chlorine attack due to a large amount of end amine groups for N-chlorination . Therefo2SO4 rejection were used to evaluate the chlorine resistances of the fabricated NF membranes. As shown in 2SO4 rejection decreased with the increasing chlorine exposure time and displayed the opposite order: TFC-PEI-blank < TFN-PEI-GO-20 < TFN-PEI-GO-40. These results indicated that the incorporation of GO in the active layer contributed to improving the chlorine resistance of the PEI-based PA NF membranes. On the one hand, the skeleton structure of GO could absorb chlorine radicals to form O–Cl bonds, accordingly reducing the chlorine radical attack toward the PA layer [The normalized water flux and the normalized NaPA layer . On the PA layer . Thus, t−2·h−1 without sacrificing salt rejection, which was 36.8% higher than the TFC-PEI-blank membrane. Meanwhile, the positively charged TFC-PEI-blank and TFN-PEI-GO membranes presented superior rejections for heavy metal ions  due to the synergistic effects of Donnan exclusion and size exclusion. Furthermore, both the TFC-PEI-blank and TFN-PEI-GO-20 membranes exhibited excellent antifouling performance toward the cationic surfactants and basic protein (LYZ), owing to the hydrophilic, smooth, and positively charged surface. Finally, the TFN-PEI-GO membranes presented the enhanced chlorine resistances due to the extra protection from GO. Therefore, the positively charged PEI-based TFN NF membrane embedded with GO shows a promising potential for wastewater purification from heavy metal ions.To construct the positively charged NF membrane with enhanced performance, the PEI-based TFN NF membrane using PEI as the aqueous reactive monomers was fabricated by embedding GO into the active layer via the IP process. After incorporating an appropriate amount of GO into the PEI-based PA active layer, the optimal TFN-PEI-GO-20 membrane showed the maximal water flux of 70.3 L·m


Novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) became pandemic by the end of March 2020. In contrast to the 2002–2003 SARS-CoV outbreak, which had a higher pathogenicity and lead to higher mortality rates, SARSCoV-2 infection appears to be much more contagious. Moreover, many SARS-CoV-2 infected patients are reported to develop low-titer neutralizing antibody and usually suffer prolonged illness, suggesting a more effective SARS-CoV-2 immune surveillance evasion than SARS-CoV. This paper summarizes the current state of art about the differences and similarities between the pathogenesis of the two coronaviruses, focusing on receptor binding domain, host cell entry and protease activation. Such differences may provide insight into possible intervention strategies to fight the pandemic. SARS-CoV-2 rapidly became pandemic by the end of March 2020, forcing much of the world to adopt lockdown strategies and putting health care systems under pressure while major concern about global health and economic stability arose. In contrast to the 2002–2003 SARS-CoV outbreak, which had a higher pathogenicity and lead to higher mortality rates, SARS-CoV-2 infection appears to be much more contagious, rapidly spreading to all continents. Compared to SARS-CoV, SARS-CoV-2 infection is characterized by a wider clinical spectrum, including asymptomatic infection, mild upper respiratory tract illness, severe viral pneumonia with respiratory failure and death –3128–31]Both SARS-CoV and SARS-CoV-2 use human ACE2 as entry receptor and human proteases as entry activators. In vitro studies have identified strategies that SARS-CoV-2 adopts to infect human cells that potentially contribute to wide spread of the virus and to immune evasion. These cell entry mechanisms may represent substantial target for host immune surveillance and provide insight into possible intervention strategies to fight the pandemic induced by this novel agent.


Glycine max), as an important oilseed crop, is constantly threatened by abiotic stress, including that caused by salinity and drought. bZIP transcription factors (TFs) are one of the largest TF families and have been shown to be associated with various environmental-stress tolerances among species; however, their function in abiotic-stress response in soybean remains poorly understood. Here, we characterized the roles of soybean transcription factor GmbZIP15 in response to abiotic stresses. The transcript level of GmbZIP15 was suppressed under salt- and drought-stress conditions. Overexpression of GmbZIP15 in soybean resulted in hypersensitivity to abiotic stress compared with wild-type (WT) plants, which was associated with lower transcript levels of stress-responsive genes involved in both abscisic acid (ABA)-dependent and ABA-independent pathways, defective stomatal aperture regulation, and reduced antioxidant enzyme activities. Furthermore, plants expressing a functional repressor form of GmbZIP15 exhibited drought-stress resistance similar to WT. RNA-seq and qRT-PCR analyses revealed that GmbZIP15 positively regulates GmSAHH1 expression and negatively regulates GmWRKY12 and GmABF1 expression in response to abiotic stress. Overall, these data indicate that GmbZIP15 functions as a negative regulator in response to salt and drought stresses.Soybean ( As a result of their sessile nature, plants are subject to variable biotic and abiotic-stress conditions. As the most pertinent abiotic-stress conditions, drought and salinity threaten the growth and productivity of crops. Plants respond and adapt to these stress conditions by activating stress-related pathways, which comprise signal perception and transduction, regulation of gene expression, and biochemical and physiological responses . Signali2O2) and superoxide (O2−); however, ROS overaccumulation is cytotoxic .Leaves of 2-week-old soybean plants including WT and


The formation of contacts among protein secondary structure elements (SSEs) is an important step in protein folding as it determines topology of protein tertiary structure; hence, inferring inter-SSE contacts is crucial to protein structure prediction. One of the existing strategies infers inter-SSE contacts directly from the predicted possibilities of inter-residue contacts without any preprocessing, and thus suffers from the excessive noises existing in the predicted inter-residue contacts. Another strategy defines SSEs based on protein secondary structure prediction first, and then judges whether each candidate SSE pair could form contact or not. However, it is difficult to accurately determine boundary of SSEs due to the errors in secondary structure prediction. The incorrectly-deduced SSEs definitely hinder subsequent prediction of the contacts among them.We here report an accurate approach to infer the inter-SSE contacts  using the deep object detection technique. The design of ISSEC is based on the observation that, in the inter-residue contact map, the contacting SSEs usually form rectangle regions with characteristic patterns. Therefore, ISSEC infers inter-SSE contacts through detecting such rectangle regions. Unlike the existing approach directly using the predicted probabilities of inter-residue contact, ISSEC applies the deep convolution technique to extract high-level features from the inter-residue contacts. More importantly, ISSEC does not rely on the pre-defined SSEs. Instead, ISSEC enumerates multiple candidate rectangle regions in the predicted inter-residue contact map, and for each region, ISSEC calculates a confidence score to measure whether it has characteristic patterns or not. ISSEC employs greedy strategy to select non-overlapping regions with high confidence score, and finally infers inter-SSE contacts according to these regions.Comprehensive experimental results suggested that ISSEC outperformed the state-of-the-art approaches in predicting inter-SSE contacts. We further demonstrated the successful applications of ISSEC to improve prediction of both inter-residue contacts and tertiary structure as well. The experimental technologies to resolve protein tertiary structures, such as X-ray crystallography, NMR spectroscopy, and cryo-electron microscopy, have achieved great successes; however, these technologies are usually time-consuming. Thus, predicting protein structure from amino acid sequence is of great importance [Proteins play important roles in a large variety of biological processes. The biological roles of proteins are mainly determined by their three dimensional structures  to the prediction of inter-SSE contacts. ISSEC is rooted in the observation that, in the inter-residue contact map, the contacting SSEs usually form a rectangle region with characteristic patterns Fig. : Fig. S1ISSEC applies the object detection technique to detect the rectangle regions with characteristic patterns, and infers inter-SSE contacts based on these regions. ISSEC has the following advantages: (1) It uses deep convolution to extract high-level features from the predicted inter-residue contacts and thus could tolerate excessive noises in these contacts. (2) ISSEC examines multiple overlapping regions in the predicted contact map and thus could get rid of the dependency on the pre-defined SSEs. (3) ISSEC could predict multiple types of inter-SSE contact simultaneously. Experimental results on PSICOV , CASP11 We first explain the concept of ISSEC using protein 3a4tA as a concrete example. Next we show the prediction accuracy of ISSEC on a variety of datasets, including PSICOV dataset, TEST1000 dataset, and transmembrane proteins. Finally, we show the application of ISSEC to improve inter-residue prediction and tertiary structure prediction as well.From the proteins in PDB25 , we randomly selected 1,000 proteins to construct a test set called TEST1000. The other test sets include: (1) PSICOV118 dataset: consisting of 118 proteins (after excluding 32 proteins without inter-SSE contacts from PSICOV dataset). (2) Mem30 dataset: consisting of 30 transmembrane proteins obtained from MemConP . (4) Betcontacts .The training set was also constructed based on PDB25. To guarantee low sequence identity between training and testing set, we excluded the proteins with sequence identify over 25% with any protein in the test sets and finally obtained a training set with 9241 proteins.E represents a H represents an The protein 3a4tA is an entclass1pt{minimaFor this protein, ISSEC first predicted probabilities of inter-residue contacts . The loL contacts), CASP11 dataset (3.4% improvement for top L contacts) and CASP13 dataset (2.0% improvement for top L/10 contacts). It was well recognized that the prediction of long-range contacts is a challenging task. This result clearly suggested that the global structure information identified by ISSEC could greatly facilitate accurate prediction of inter-residue contacts.As illustrated in Additional file When inter-SSE contacts are known, topology of the full tertiary structures are almost fixed. Here, we used the predicted inter-SSE contacts to guide structure building. Specifically, we integrated ISSEC into CONFOLD  as folloFigure Our ISSEC could currently predict three frequent types of inter-SSE contacts, i.e., In this study, we present an approach to predicting inter-SSE contacts. Experimental results suggested that this approach could be used to predict ISSEC infers inter-SSE contacts through detecting rectangle regions with characteristic patterns in the inter-residue contact map. To detect these rectangle regions, ISSEC employs the object detection framework, which was initially proposed for detecting objects in images . For exaThe similarity between inter-residue contact maps and images enables us to apply the object detection technique to infer inter-SSE contacts. Specifically, ISSEC takes the predicted inter-residue contact map as input, and outputs a group of objects representing contacting SSEs Fig. . For eacmentclass2pt{minimExtracting high-level features: To overcome the drawback of directly using the predicted probabilities of inter-residue contact, ISSEC extracts high-level features using a feature pyramid network . FPN usProposing candidate rectangle regions: As we have no knowledge of location or size of the rectangle regions formed by contacting SSEs in advance, we propose multiple candidate rectangle regions and expect at least one of these proposed regions to cover the contacting SSEs. ISSEC accomplishes this objective using a region proposal network  Tracing back to the input inter-residue contact map: From each rectangle region on the feature map, ISSEC traces back to a larger rectangle region on the input inter-residue contact map. This tracing back operation is accomplished using ROIAlign [rk , which ROIAlign .It is worthy pointing out that the rectangle regions formed by contacting SSEs are relatively small; more specifically, each of these regions does not exceed 1/4 of the input inter-residue contact map. This observation provides the possibility to cover all contacting SSEs using the proposed candidate rectangle regions. ISSEC achieves this objective through appropriately choosing various shape and size of the rectangle regions on the feature maps  as shown in Additional file  as showngreedy selection strategy that works as follows: We first filter out the rectangle regions that overlap with diagonal and the regions whose confidence score less than a threshold (selecting and removing step is repeated until all candidate rectangle regions were processed.To identify non-overlapping rectangle regions, ISSEC uses a For each of the selected non-overlapping rectangle regions, ISSEC reports the corresponding inter-SSE contact type, position, and confidence score as final results. Finally, a rectangle region that had > 80% overlap with a native contact was correct one.However, despite the similarities between inter-residue contact maps and images, the contacting SSEs differ greatly from the objects in images. To fit in the specific properties of inter-SSE contacts, we modified the generic object detection framework. Figure In the training process, ISSEC uses the multi-task loss, including classification loss, localization loss and mask loss (further details in Additional file Unlike the Additional file 1: Supplementary methods, tables and figures.


A and glycine receptors, reverse chronic pain-related behaviors in preclinical assays. Collectively, these evidences strongly suggest the pharmacological modulation of pentameric ligand-gated ion channels is a promising strategy towards the development of novel therapeutics to treat chronic pain states in humans.Chronic pain is a common detrimental condition that affects around 20% of the world population. The current drugs to treat chronic pain states, especially neuropathic pain, have a limited clinical efficiency and present significant adverse effects that complicates their regular use. Recent studies have proposed new therapeutic strategies focused on the pharmacological modulation of G-protein-coupled receptors, transporters, enzymes, and ion channels expressed on the nociceptive pathways. The present work intends to summarize recent advances on the pharmacological modulation of pentameric ligand-gated ion channels, which plays a key role in pain processing. Experimental data have shown that novel allosteric modulators targeting the excitatory nicotinic acetylcholine receptor, as well as the inhibitory GABA Chronic pain is defined as pain that persists after a normal healing time . ChronicThe current pharmacological therapeutics to manage chronic pain mainly includes non-opioid analgesics and opioids [reviewed in ]. Weak oA major issue of the long-term use of both non-opioid and opioid analgesics is that pain relief is often achieved at the expense of unwanted adverse events (AEs) . ConstipIn addition to the AEs described above, the clinical efficacy of the current treatments against chronic pain, particularly those directed to neuropathic pain, is significantly limited . This sc3R), γ-aminobutyric type A (GABAAR), and glycine receptors (GlyR) (Pentameric ligand-gated ion channels (pLGICs), a family of channels previously known as Cys-loop receptors, are main players of the chemical neurotransmission on the central nervous system . Mammalis (GlyR) . pLGICs s (GlyR) Figure 1s (GlyR) . The bins (GlyR) . The strs (GlyR) .ARs, and GlyRs display analgesic effects in behavioral models of chronic pain  displayed effective pain management on intestinal bowel syndrome (5-HT systems . The effsyndrome  and fibrsyndrome .50 ≈3 μM) by using two-electrode voltage-clamp (TEVC) recordings in Xenopus oocytes. Interestingly, compound 111 exerted analgesic activity in CFA-injected rats  as an antagonist for both human and rodent α9α10nAChRs . However, a large proportion of GABAARs are composed by two α-subunits, two β-subunits, and one γ-subunit , attenuate nociceptive transmission in animal models of chronic pain , a non-sedative analog of propofol . 2,6-DTBin vitro/in vivo laboratory experiments to human clinical trials is still a critical issue. The recent development of new stem-cells and gene editing technologies may offer a viable alternative for the study of allosteric modulators using neurons derived from human-induced pluripotent stem cells (hiPSC) of specific patients , FONDECYT 1161014 (to MC), FONDECYT 3170108 (to CB), and VRID 219.033.111-INV (to GM-C). CL was supported by CONICYT doctoral fellowship 21171549.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.


For patients with at least a complete response, to reach double negativity (FDG-PET/CT and MRD) is a predictive surrogate for patient outcome. Homogenization of FDG-PET/CT interpretation after therapy, especially clarification of complete metabolic response definition, is currently underway. FDG-PET/CT does not allow MRD to be evaluated when it is negative at initial workup of symptomatic MM. New PET tracers such as CXCR4 ligands have shown high diagnostic value and could replace FDG in this setting. New sensitive functional magnetic resonance imaging (MRI) techniques such as diffusion-weighted MRI appear to be complementary to FDG-PET/CT for imaging MRD detection. The goal of this review is to examine the feasibility of functional imaging, especially FDG-PET/CT, for therapeutic assessment and MRD detection in MM.Serum markers and bone marrow examination are commonly used for monitoring therapy response in multiple myeloma (MM), but this fails to identify minimal residual disease (MRD), which frequently persists after therapy even in complete response patients, and extra-medullary disease escape. Positron emission tomography with computed tomography using 18F-deoxyglucose (FDG-PET/CT) is the reference imaging technique for therapeutic assessment and MRD detection in MM. To date, all large prospective cohort studies of transplant-eligible newly diagnosed MM patients have shown a strong and independent pejorative prognostic impact of not obtaining complete metabolic response by FDG-PET/CT after therapy, especially before maintenance. The FDG-PET/CT and MRD (evaluated by flow cytometry or next-generation sequencing at 10 Multiple myeloma (MM) is a plasma cell malignancy characterized by a clonal population of bone marrow plasma cells that secrete a monoclonal paraprotein or an immunoglobulin free light chain. Whilst the detection of MM can result from a broad range of indications, including anemia, hypercalcemia, and renal failure, the most common symptom is lytic bone disease owing to an over-activation of osteoclasts, and occurs in over 80% of patients . ImagingNovel and effective agent-based therapies have led to significant improvements in the treatment of MM patients, with many achieving a profound biological response. This is a crucial goal for transplant-eligible newly diagnosed MM patients, because of its major prognostic impact. Whilst serum markers and bone marrow examination is routinely used for monitoring response to therapy in MM , it failMM induces a high spatial genomic heterogeneity, resulting in the cohabitation of distinct disease clones within a patient that exhibit various genomic profiles in the bone marrow and in bone focal lesions . MoreoveFDG-PET/CT is the gold-standard imaging technique for therapeutic assessment in MM ,6. FDG-PThe great strength of FDG-PET/CT after therapy for MM is that it allows the detection of residual active clonal plasma cells within residual lytic bone lesions. These commonly persist for a long time after the start of therapy. As shown in Conversely, sclerotic changes in lytic lesions such as traditional callus formation around rib lesions or sclerotic rim development around axial and peripheral skeleton lesions can lead to the misinterpretation of FDG-PET/CT images and result in false-positive post-therapy images.Since the seminal study published by Bartel et al. in 2009  examinedmax, traducing intensity of carbohydrate metabolism) of residual focal bone lesions > 4.2 had a significantly lower progression-free survival. Secondly, patients were assessed by FDG-PET/CT imaging 3 months after autologous stem cell transplantation. Complete metabolic response, defined similarly to Bartel et al. as the complete absence of residual uptake of the initial targets, was reached for 65% of patients. Progression-free and overall survival of these patients at 4 years was significantly higher than for the PET-positive patients . Moreover, 23% of all patients who achieved complete biological response according to the IMWG’s regular guidelines harbored an FDG-PET/CT-positive lesion and were associated with poorer outcome, meaning residual FDG uptake after therapy could detect MRD. Multivariate analysis of all statistically significant prognostic variables by univariate analysis confirmed that FDG-PET/CT status after autologous stem cell transplantation (before maintenance therapy) was one of the most significant independent predictors of progression-free survival. Afterwards, in 2015, the same group reproduced these results in a cohort of 282 symptomatic MM patients initially treated between 2002 and 2012 [max of focal bone lesions >4.2), an international staging system > 2 and a failure to reach complete biological response. These three features were then combined to build a prognostic scoring system. After therapy, FDG-PET/CT negativity (defined above) was reached in 70% of patients, while only 53% percent of patients reached a conventional biological complete response. Once again, FDG-PET/CT negativity after therapy (before maintenance) clearly positively influenced progression-free and overall survival of patients. FDG-PET/CT negativity was independently correlated with extended progression-free and overall survival, even in the biological complete response patient sub-group. Of the sixty-three percent of patients who relapsed or progressed after first-line therapy, bone progression was identified exclusively by systematic FDG-PET/CT in 12% of them. Residual focal bone lesions uptake of SUVmax >4.2 after first-line therapy was independently correlated with exclusive FDG-PET/CT progression.In 2011, Zamagni et al. explored the prognostic relevance of FDG-PET/CT after thalidomide-dexamethasone induction therapy and double autologous stem cell transplantation in 192 newly diagnosed MM patients . Firstlyand 2012 . FDG-PETMore recently, the prospective multicenter French IMAJEM trial  aimed tomax of the initial target (ΔSUVmax) was the strongest independent prognostic factor regarding to hazard ratio, which was the more discriminative, especially as compared to conventional biological response. Patients with a reduction in uptake of more than 25% after three cycles of induction therapy had a significantly improved progression-free survival.A second analysis of the IMAJEM cohort that only considered patients with baseline FDG-avid focal bone lesions and/or bone marrow involvement  corroborated the powerful prognostic value of FDG-PET/CT results after three cycles of induction therapy . Indeed,max, the shorter the time to relapse. Among patients who did not relapse during follow-up, 27 had a negative post-therapy PET, whereas 13 had a positive post-therapy PET. None of the patients who did not relapse had an SUVmax progressive increase during the follow-up.Using a smaller prospective cohort of 107 MM patients, Nanni and colleagues also analyzed the prognostic impact of FDG-PET/CT after therapy . PatientThe open issue concerning FDG-PET/CT interpretation after therapy, notably before maintenance, is to find out the uptake cut-off of residual lesions corresponding to complete metabolic response definition. Indeed, in the trials summarized above, complete metabolic response was not similarly defined , and a h−5 and 10−6, respectively. Concordance of the PET/CT and MRD results in ≥CR patients (PET-CR/MRD) identified 60 and 28 PET-CR-negative patients at 10−5 and 10−6 levels, respectively. Pre-maintenance double negativity rates for PET/CT and MRD were higher in D-VTd-treated patients compared to the Vtd patients . Because PET and MRD results are complementary, PET-CR/MRD negativity concordance may provide insight into using both methods as a predictive surrogate for patient outcome.Finally, in the large international CASSIOPET study, a companion study of CASSIOPEIA , pre-maiRasche et al. recently published a study that examined the FDG-PET/CT false-negative rate within a cohort of 227 newly diagnosed MM patients, and identified the tumor-intrinsic parameters associated with this pattern . Whilst Thus, FDG-PET/CT is not suitable for MM therapeutic assessment and MRD detection in this sub-group of baseline false-negative FDG-PET patients.68Ga-Pentixafor ligand targeting CXCR4 up until now, here we restrict our review to the potential interest of this CXCR4 tracer for therapeutic assessment and MRD detection in MM.The growth in molecular imaging developments is highly relevant for MM imaging. Novel PET tracers aiming to target different metabolic pathways or plasma cell receptors have demonstrated interesting preliminarily results for disease detection and could be used for MRD detection, especially in FDG-PET/CT negative patients. These include methionine, which is an amino-acid PET tracer, lipid tracers such as choline or acetate, and other promising immuno-PET targets such as CD138 and CD38. Because we recently reviewed several of these novel tracers , and bec68Ga-Pentixafor, a labelled peptide with high affinity for CXCR4 and an excellent signal-to-noise ratio in CXCR4-expressing patients is a promising PET ligand [177lutetium or 90yttrium. Initial CXCR4-directed endo-radiotherapy with 177Lu- and 90Y-labeled pentixather [CXCR4 is a G-protein-coupled chemokine receptor family member . CXCL12 T ligand ,27,28, etixather  for progThere are still discrepancies between FDG and CXCR4 tracers with respect to disease detection. FDG remains more sensitive than CXCR4 for a large proportion of cases. Indeed, receptor expression on the monoclonal plasma cell surface appears to be an active process that could be deeply impacted by a preceding or concomitant chemotherapy , and fur−2 [−2 is commonly chosen. DW-MRI produces high quality image contrast between normal and diseased bone marrow, especially compared to conventional morphological sequences [Conventional morphological MRI imaging is not a reliable tool for the therapeutic assessment of MM because focal bone abnormalities can persist a long time after the start of therapy without any vital cells, resulting in a high false-positive imaging rate during follow-up . In the −2 . Howeverequences ,33,34. Bequences ,36,37.In MM, ADC percent changes seem to be significantly higher in good responder patients  compared to patients without a profound response  . ChangesThe potential of DW-MRI for MRD detection in MM after therapy has also been investigated  in a stuep (reflecting vessel permeability and perfusion) reflect bone marrow angiogenesis/microcirculation, which plays a central role in the pathogenesis of MM, notably because it is correlated to MM-related bone disease and because of its pejorative prognostic significance [Dynamic contrast-enhanced MRI (DCE-MRI) is a dynamic study of the accumulation and distribution of contrast medium (Gadolinium-DTPA) in the region of interest (in T1-weighted sequence) after a constant pump-controlled bolus injection. DCE-MRI-derived quantitative parameters such as amplitude A (correlated with blood volume) and exchange rate constant kificance ,43,44. Iificance ,46.−5 and 10−6 levels respectively) results are complementary for MRD detection outside and inside the bone marrow. For patients with at least a biological complete response, achieving double negativity (FDG-PET/CT and intra-medullary MRD) is a predictive surrogate for patient outcome and a trustworthy and premature biomarker of treatment effectiveness.FDG-PET/CT is the preferred imaging technique for the therapeutic assessment of MM. All large prospective studies of transplant-eligible newly diagnosed MM patient cohorts showed a strong and independent pejorative prognostic impact of not achieving complete metabolic response by FDG-PET/CT after therapy, especially before maintenance therapy. FDG-PET/CT and MRD (evaluated by multi-parametric flow cytometry or next generation sequencing at 10However, FDG-PET/CT is not yet widely used in this setting in clinical practice. In order to extend its purpose, open issues need to be addressed, especially homogenization of interpretation guidelines for reporting results. Because the definition of complete metabolic response in FDG-PET/CT is not clearly established, the final results of prospective analyses currently underway, especially the CASSIOPET study, are expected to standardize the interpretation criteria of FDG-PET/CT after therapy to better define a cut-off to determine PET positivity/negativity for MRD assessment outside the bone marrow.FDG-PET/CT is not suitable for MM therapeutic assessment and MRD detection in a sub-group of baseline symptomatic false-negative FDG-PET/CT patients. Novel PET tracers aiming to target different metabolic pathways or plasma cell receptors like CXCR4 are being employed in current studies and are expected to alleviate the known limitations of FDG. Whilst new sensitive functional MRI sequences such as DW-MRI are not routinely used in the clinic, its use in experimental research settings suggests that it will be very useful in complementing FDG-PET/CT imaging for MRD detection. DW-MRI needs to be further studied and correlated with clinical outcomes so simultaneous acquisition with a PET-MRI systemsounds attractive. PET-MRI will allow homogeneous prospective therapeutic assessment comparisons between FDG-PET/CT and DCE-MRI as well, particularly by comparing metabolic and bone marrow angiogenesis changes.Finally, the influence of MRD assessment on treatment approaches still has to be defined.


Clinical manifestations associated with covid-19 seem to be not limited to the lungs and widen to affect several organs including skin. Through two cases well documented, we report cutaneous manifestations strongly suggestive of Covid-19 which dermatologists are beginning to report worldwide and which we proposed to call them polymorphous acral eruption. Case 1: a 17-year-old adolescent with no medical history, especially chilblains, had asymptomatic erythematopurpuric lesions of the toes and soles for four days (our days . There wCase 2: a 23-year-old young woman, with no pathological history, presented for a week papulovesicular lesions of the fingers without pain or pruritus (pruritus , concomiSince description of Covid-19, we are witnessing every day new clinical observations and new affected organs; skin is one of them, despite Darlenski and Tsankov ruling out a cutaneous manifestation of covid-19 . Hoenig Curiously, several dermatologists have noticed acral eruptions like chilblains , purpuraThe authors declare no competing interests.


CD123 is overexpressed in multiple hematologic malignancies. Advances in CD123-targeted therapies over the past decade have positioned this molecule as an integral biomarker in current practice. This review provides an overview of CD123 biology and in-depth discussion of clinical laboratory techniques used to determine CD123 expression in various hematolymphoid neoplasms. In addition, we describe various pharmacologic strategies and agents that are available or under evaluation for targeting CD123.CD123, the α chain of the interleukin 3 receptor, is a cytokine receptor that is overexpressed in multiple hematolymphoid neoplasms, including acute myeloid leukemia, blastic plasmacytoid dendritic cell neoplasm, acute lymphoblastic leukemia, hairy cell leukemia, and systemic mastocytosis. Importantly, CD123 expression is upregulated in leukemic stem cells relative to non-neoplastic hematopoietic stem cells, which makes it a useful diagnostic and therapeutic biomarker in hematologic malignancies. Varying levels of evidence have shown that CD123-targeted therapy represents a promising therapeutic approach in several cancers. Tagraxofusp, an anti-CD123 antibody conjugated to a diphtheria toxin, has been approved for use in patients with blastic plasmacytoid dendritic cell neoplasm. Multiple clinical trials are investigating the use of various CD123-targeting agents, including chimeric antigen receptor-modified T cells (expressing CD123, monoclonal antibodies, combined CD3-CD123 dual-affinity retargeting antibody therapy, recombinant fusion proteins, and CD123-engager T cells. In this review, we provide an overview of laboratory techniques used to evaluate and monitor CD123 expression, describe the strengths and limitations of detecting this biomarker in guiding therapy decisions, and provide an overview of the pharmacologic principles and strategies used in CD123-targeted therapies. CD123 has emerged in recent years as an attractive novel target of therapy. CD123 is normally expressed on hematopoietic stem cells (HSC) . Indeed,Efforts to target CD123 have resulted in the development of agents that effectively target neoplastic cells expressing this molecule. Several drugs designs have been explored in furtherance of this goal, mostly utilizing a humanized anti-CD123 antibody moiety fused with a toxic payload. In parallel, cellular therapy approaches using CD123-targeting chimeric antigen receptor (CAR) T cells have emerged as another exciting approach ,7. As thIn this review, we provide an overview of CD123 biology, discuss current clinical laboratory techniques used to evaluate CD123 expression on neoplastic cells and salient features of CD123 expression in various hematolymphoid neoplasms, and discuss the major classes of targeted CD123-targeting agents available at present.c) family of receptors, which also includes IL-5R and granulocyte-monocyte colony stimulating factor (GM-CSF) receptor. This family of membrane receptors regulates the growth, proliferation, survival, and differentiation of hematopoietic cells, along with immunity and inflammatory response  ,54. NotaThe expression of CD123 on mature B lymphoproliferative disorders in general and B-cell disorders with hairy or villous lymphocytes in particular was extensively investigated. CD123 overexpression has been demonstrated in almost all cases of HCL, with rare expression in HCL variant and splenic lymphoma with villous lymphocytes . Recent CD123 is expressed on Hodgkin–Reed–Sternberg cells in 90% of Hodgkin lymphoma (HL), and IL-3 is capable of inducing Hodgkin cell line growth in vitro . CD123 oA number of CD123-targeted therapies have been developed over the past decades. The ensuing section is aimed at providing an overview of design strategies and salient advances in the use of CD123-targeted therapies in patients with various hematolymphoid malignancies. In 2000, Frankel and colleagues  reportedAn ongoing clinical trial (NCT02268253) is evaluating the effect of tagraxofusp in patients with myelofibrosis . PrelimiFrolova et al.  confirmeThe interaction between plasma cell myeloma cells and pDCs stimulate IL-3 expression, which consequently enhances myeloma cells’ development and increases pDC survival ,68. TargA phase I/II study NCT00397579) evaluated the response of 11 patients with BPDCN who received tagraxofusp. Five patients had CR and two had partial response after a single course; the duration of clinical response fluctuated between 1 and 20 months  . Similar79 evaluaJin and colleagues  reportedAfter chemotherapy and transplantation, the number of NK cells is increased in AML patients , furtherAlthough HL cells express CD123, the limited number of NK cells in the microenvironment limits the ADCC induced by talacotuzumab. Ernst and colleagues demonstrated that the combination of CSL362 and high-affinity Fc gamma-receptor NK-92 cells (haNK) effectively target and kill HL cells in vitro . Based oH domain of one antibody and the VL of the other, recognizing the N-extracellular domain of CD123 and the extracellular domain of CD3 within the human T cell receptor complex. [Kuo and colleagues  developecomplex.  The drugcomplex. . Due to complex. .Godwin et al.  studied APVO436 consists of two full-length IgG bispecific immunoglobulins that retain the Fc region of the antibody and therefore has higher stability in vivo and a longer half-life compared to DART, thus requiring intermittent instead of continuous infusion (NCT03647800) .JNJ-63709178 is being assessed for the treatment of RR AML (NCT02715011), and XmAb14045 is being studied for the treatment of AML, B-ALL, BPDCN, and CML blast crisis (NCT02730312). Other bi-specific antibodies have been designed to include a novel conjugate of single-chain Fv antibody fragments specific for CD123 with an anti-CD16 antibody to enhance the recruitment of CD16-positive NK cells, thereby inducing better ADCC activity .Recently, triplebody dual-targeting molecules have been developed using triple single-chain Fv antibody fragments (scFvs). These molecules have longer half-lives and can target both CD33/CD123 positive tumor cells as well as CD16 on NK cells, inducing a stronger elimination of leukemic cells in vitro . BraciakThe deletion of eight C-terminal amino acid residues from IL-3 engender higher affinity interactions with its receptor CD123 and better anti-leukemic effect in drug constructs . Fan andThis product consists of two single chain variable fragments (scFVs) specific for tumor antigen and for CD3. This construct provides a unique characteristic to destroy tumor cells and attract resident T cells to neoplastic cells. The infusion of genetically modified T cells will allow an accumulation of their recombinant bispecific antibody product in the tumor area, hence prolonging the treatment effect. The engager had significant in vivo cytotoxic activity against tumor cells and notable prolongation in survival . CD123-EIMGN632 is a CD123-targeting antibody-drug conjugate (ADC) consisting of a humanized anti-CD123 antibody linked to a DNA mono-alkylating cytotoxic payload known as indolinobenzodiazepine pseudodimer. This molecule was tested in vivo and in vitro showing limited toxicity in normal myeloid progenitors . A phaseAngelova and colleagues showed that IMGN632 therapy in vitro was effective against B-ALL cell lines and primary B-ALL cells expressing CD123, with no effect on normal lymphocytes . CurrentA CAR is a fusion protein combining an extracellular domain that consists of a single-chain variable fragment (scFv) derived from antibodies, a trans-membrane connecting segment, and an intra-cellular signaling domain. The intracellular domain of first-generation CARs consisted only of the zeta-signal-transducing subunit of the TCR/CD3 receptor complex (CD3z), whereas second- and third-generation CARs possess respectively one or two co-stimulatory endo-domain molecules, such as CD28, OX40, and CD137 fused to CD3z . CAR T cCD123 is an attractive target for CART in AML; however, since CD123 is dimly expressed on some normal hematopoietic cells and endothelial cells, undesirable off-target effects can result . In addiA phase I clinical trial (NCT02159495) is examining the safety and efficacy of CD123 CAR T cell therapy using a lentivirally transduced second-generation CAR with CD28 co-stimulation (CD123CAR-CD28-CD3ζ-EGFRt) in adult patients with relapsed/refractory AML and persistent or recurrent BPDCN. Clinical remission has been achieved in patients treated with low doses of CD123 CART (MB-102) without evidence of dose-limiting side effects . Based o4 with sparing of normal hematopoietic stem cells that express lower antigen densities [Furthermore, a CAR lentiviral vector containing the CD123CAR-CD28-CD3ζ-EGFRt was designed with CD123+ hematopoietic stem cells in MDS. CAR T cells were able to attack CD123+ cells with an antigen density of >1 × 10ensities .The applications of CD123CART have expanded to encompass treatment-resistant HL cases. Ruella et al. demonstrated that the CD123 CART could overcome the immunosuppressive tumor microenvironment of HL and eliminate the HC .The tremendous advances achieved recently in understanding of cancer pathogenesis allowed the emergence of new biomarkers that can have significant diagnostic, prognostic, and therapeutic implications. CD123 has gained particular attention due to its widespread expression on numerous hematolymphoid malignancies, particularly AML and BPDCN. Promising CD123 targeted therapies have been developed and continue to be examined, ensuring the continued need to evaluate CD123 expression as an integral biomarker whose presence represents a targetable vulnerability.


The worldwide struggle against the coronavirus disease 2019 (COVID-19) as a public health crisis continues to sweep across the globe. Up to now, effective antiviral treatment against COVID-19 is not available. Therefore, throughout virus infections, a thorough clarification of the virus-host immune system interactions will be most probably helpful to encounter these challenges. Emerging evidence suggests that just like SARS and MERS, COVID-19 primarily suppresses the innate immune system, enabling its stable propagation during the early stage of infection. Consequently, proinflammatory cytokines and chemokines have been increasing during infection progression associated with severe lung pathology. It is imperative to consider hyper inflammation in vaccine designing, as vaccine-induced immune responses must have a protective role against infection without leading to immunopathology. Among the front-line responders to viral infections, Natural Killer (NK) cells have immense therapeutic potential, forming a bridge between innate and adaptive responses. A subset of NK cells exhibits putatively increased effector functions against viruses following pathogen-specific and immunization. Memory NK cells have higher cytotoxicity and effector activity, compared with the conventional NK cells. As a pioneering strategy, prompt accumulation and long‐term maintenance of these memory NK cells could be an efficacious viral treatment. According to the high prevalence of human cytomegalovirus (HCMV) infection in the world, it remains to be determined whether HCMV adaptive NK cells could play a protective role against this new emerging virus. In addition, the new adaptive-like KIR+NKG2C+ NK cell subset (the adaptive-like lung tissue residue [tr]NK cell) in the context of the respiratory infection at this site could specifically exhibit the expansion upon COVID-19. Another aspect of NK cells we should note, utilizing modified NK cells such as allogeneic off-the-shelf CAR-NK cells as a state-of-the-art strategy for the treatment of COVID-19. In this line, we speculate introducing NKG2C into chimeric antigen receptors in NK cells might be a potential approach in future viral immunotherapy for emerging viruses. In this contribution, we will briefly discuss the current status and future perspective of NK cells, which provide to successfully exploit NK cell-mediated antiviral activity that may offer important new tools in COVID-19 treatment. World Health Organization declared the outbreak of coronavirus disease 2019 (COVID-19) a Public Health concern on 30 January 2020 . This noIn this regard, Natural Killer (NK) cells as essential front-line responders to many viral infections in humans have been proposed for a suitable therapeutic approach in severe COVID-19 patients, and several clinical trials have begun . In thisCoronaviruses(CoVs) belong to the Coronaviridae family and are characterized by a positive-sense single-strand ribonucleic acid (RNA). The coronavirus genome is highly susceptible to mutations that result in genetic drift and evade immune recognition . ConsistAccumulating evidence suggests that the stimulated immune response by SARS-CoV-2 infection results in two phases; first, immune defense-based protective phase at the early stage, and second, severe-stage inflammation-driven damaging phase . Once thThe immunomodulatory strategies that are used by beta coronaviruses can lead to dysregulated IFN responses . SimilarThus, it is essential to diagnose the infection during the first phase before the cytokine storm is initiated . In addiEvaluation of diagnostic accuracy for COVID-19 is still in progress, so an authentic interpretation of the findings in this area is essential in guiding patient care. Indeed, clear pattern of inflammatory and immune biomarker abnormalities is needed to discriminate the stages of infection, which may potentially aid in addition to molecular detection of virus. The detection of both SARS-CoV-2 nucleic acid and specific antibodies to viral proteins have thus far become significant for primary diagnosis infection and immunity in COVID-19 patients, respectively. Two types of reliable diagnostic tests have been thus far commonly utilized including reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoglobulin M (IgM) and IgG antibody testing . The RT-A few weeks after COVID-19 outbreak, several science groups started to design a vaccine for this novel disease. SARS-CoV and MERS-CoV immunization studies have also become a pattern for targeting antigen selection in designing vaccines . Moving NK cells are at the forefront of the antiviral response  and theyAnother mode of recognition includes expressing Fc receptors , by NK cells identifying infected cell bound to antibodies in the surface of infected target cells, called antibody-dependent cell-mediated cytotoxicity (ADCC), resulting in the release of cytotoxic factors such as perforin and proteases known as granzyme. Therefore, NK cells can also kill virus-producing cells through this important property . Virus-iThe diversity of NK cell function and phenotype is accordingly an issue of growing attractiveness. With reference to cytolytic activity, cytokine secretion, and organ-specific localization, three main subsets of NK cells can be consequently identified. Large amounts of cytokines such as IFNγ are thus produced by the CD56bright/CD16low subset in response to activation, whereas CD56low/CD16high cells are the major cytotoxic populations. Cytotoxicity and cytokine secretion are further regulated by making a balance between inhibitors and activating receptors and the effector function of NK cells also relies on the incorporation of activating and inhibitory receptors . A thirdTherapeutic strategies for COVID-19 based on the present investigations of SARS-CoV and MERS-CoV infections may be helpful in offering potential treatment targets for fighting this pandemic.M pneumonia infection and SARS patients  wasn’t significantly altered as compared to the percentages of NK cells in SARS cases , which indicates that the NK cells monitoring should be helpful in differentiating true SARS from false SARS and M pneumonia infection. Due to the greatest similarity of COVID-19 to SARS-CoV, NK cells monitoring can be investigated for this novel coronavirus (novel-CoV)  NK cells in 72 cases of severe SARS were significantly lower than those in mild cases . More imvel-CoV) . In addivel-CoV) , and theOther studies, consistent with this context, have shown severe cases of SARS patients with a lower level of NK cells than those in milder cases . LikewisRemarkably, the total number of NK cells, as well as B-cells and T-cells, can decrease significantly in patients with COVID-19 particularly in severe cases, compared to non-severe ones . In thisInterestingly, NK cells have a considerable role in inducing the lysis of virus-infected cells since the cytotoxicity of such cells is regulated by activating and inhibitory receptors. An increased NK group 2 member A (NKG2A) expression as a heterodimeric inhibitory receptor can also result in functional exhaustion of NK cells in patients with SARS-CoV-2 infection. Indeed, the total number of NK cells declines during the COVID-19 and the number of NK cells is simultaneously increased with the reduced expression of NKG2A after recovery , while tOver recent years, a novel concept has emerged indicating that the present paradigm that T-cells and B-cells are the only factors of adaptive immunity should be reconsidered. Although, NK cells have been respected for their ability to mediate spontaneous cytotoxicity during innate immune responses in an antigen-independent manner, it has become recently clear that they display long-lived immunological memory responses . In micevia ADCC in the presence of anti-HCMV antibodies. Moreover, adaptive NK cells are characterized by expression of CD57 (maturation marker), KIRs, and the absence of the inhibitory receptor NKG2A and can further produce inflammatory cytokine and cytolysis in response to target cell recognition , encompass CD56bright/CD16neg/dim NK cells, but CD56dim/CD16 bright cells settle in tissues such as lung and breast . It shouAs mentioned above, another important consideration in expanding strategies to target NK cells is related to the findings that unique tr or tissue-specific (ts) NK cell populations can display adaptive features. Recently, Marquardt et al. have identified a new adaptive-like KIR+/NKG2C+ NK cell subset with a CD49a+/CD56bright/CD16−trNK cell phenotype in human lung and in peripheral blood of HCMV-seropositive individuals, mentioned above . The adaCan SARS-CoV-2 infection specifically drive the expansion of adaptive-like trNK cells?Is disease progression in HCMV-seropositive patients involved with SARS-CoV-2 infection compared with HCMV-seronegative ones can be different? Consistent with this concept, based on clinical appearance and blood parameters during infection, we have observed that the severity of clinical manifestation in coinfection of COVID-19 and HCMV in kidney transplant patients was less severe compared with renal transplant patients with only COVID-19 (unpublished data).Is it possible to have a cross‐reactivity of HCMV-specific NK cells Figure 1A wave of investigations revealed that human NK cells presented adaptive immune responses upon immunization or infection. Therefore, NK-mediated recall responses may further allow for development of immunization-based approaches in viral treatments . NKG2C+Cversus-leukemia (GVL) reaction in patients  has produced an allogeneic and off-the-shelf cryopreserved NK cell, known as CYNK-001, derived from placental stem cells for treating leukemia and multiple myeloma   . CAR-NK (CAR-NK) . Novel s(CAR-NK)  such as (CAR-NK) , NKG2A ((CAR-NK) , and pro(CAR-NK) . Current(CAR-NK) . Recentl(CAR-NK) . In this(CAR-NK) . A recen(CAR-NK) .In conclusion, a promising approach to better management of respiratory diseases, as one of the leading causes of death worldwide, may be based on remodeling NK cells phenotypic and their functional features. It is hoped that the hypotheses addressed in this review will be employed as a stimulus for further research to help combat COVID-19 as deadly contagious disease of increasing incidence around the world. The clarification of these ideas about utilizing and modifying NK cells will further allow future dissection of potential strategies for treatment and vaccination.SS and RY conceived the idea for the manuscript. SS drafted the manuscript. RY edited and added comments into the manuscript.This study was supported by Shiraz University of Medical Science, Shiraz, Iran.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.


Pancreatic ductal adenocarcinoma (PDAC) is an extremely challenging disease with a high mortality rate and a short overall survival time. The poor prognosis can be explained by aggressive tumor growth, late diagnosis, and therapy resistance. Consistent efforts have been made focusing on early tumor detection and novel drug development. Various strategies aim at increasing target specificity or local enrichment of chemotherapeutics as well as imaging agents in tumor tissue. Aptamers have the potential to provide early detection and permit anti-cancer therapy with significantly reduced side effects. These molecules are in-vitro selected single-stranded oligonucleotides that form stable three-dimensional structures. They are capable of binding to a variety of molecular targets with high affinity and specificity. Several properties such as high binding affinity, the in vitro chemical process of selection, a variety of chemical modifications of molecular platforms for diverse function, non-immunoreactivity, modification of bioavailability, and manipulation of pharmacokinetics make aptamers attractive targets compared to conventional cell-specific ligands. To explore the potential of aptamers for early diagnosis and targeted therapy of PDAC - as single agents and in combination with radiotherapy - we summarize the generation process of aptamers and their application as biosensors, biomarker detection tools, targeted imaging tracers, and drug-delivery carriers. We are furthermore discussing the current implementation aptamers in clinical trials, their limitations and possible future utilization. Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal cancers burdened with a five-year overall survival below 9% . In 2018Aptamers have the potential to overcome difficulties of clinical diagnosis and treatment in PDAC. These molecules are small oligonucleotide sequences that serve as ligands to target molecules such as proteins, bacteria, viruses, or cells. Due to their advantages of higher tissue penetration, rapid production, low synthesis cost, less immunogenicity, thermal stability, and ease of labeling , aptamerSince 1990, Systematic Evolution of Ligands by EXponential enrichment (SELEX) became the method of choice for generating aptamers . In ordeAptamers have been used as ligands for the detection step of noninvasive diagnostic assessments, such as ELISA and other immunoassays, which are usually applied for analyzing biomarkers in blood samples. These new aptamer-based assays, termed “aptasensors” can be designed to integrate readout methods, such as chemiluminescence (CL), electro-chemoluminescence (ECL), fluorescence, surface plasmon resonance (SPR), surface-enhanced Raman spectroscopy (SERS), etc. –17, to id) value of 20.05 ± 3.02 nM showes that this aptasensor has a high affinity to CA 19–9, it has not been prospectively validated as aptasensor in biological fluids [Carbohydrate antigen 19–9 (CA 19–9), the only routinely used serum marker of PDAC, can specifically be bound by an aptamer, which has been identified using a trypsin-enhanced SELEX method. Although the dissociation constant , C4b-binding protein α-chain (C4BPA), insulin-like growth factor-binding protein 2 (IGFBP2), insulin-like growth factor-binding protein 3 (IGFBP3), interleukin-1β (IL-1β), interleukin-8 (IL-8), interleukin-10 (IL-10), vascular endothelial growth factor (VEGF), and macrophage inhibitory cytokine-1 (MIC-1) , could tThe identification of promising predictive PDAC biomarkers remains challenging. Circulating tumor cells (CTCs) are single or clustered cells that loose connection to the tumor bulk and can be detected in the bloodstream. They are important biomarkers for the diagnosis and prognosis of early and metastatic cancer . Thus, sTo date, several aptamer-based biosensors have been designed to identify and quantify various CTCs with detection limits as low as one single cell . In PDACWu and co-workers used SELEX to generate a DNA-aptamer called XQ-2d and successfully identified CD71 (transferring receptor 1) as the molecular target for cell-specific aptamer binding . Other gApart from intact PDAC cells the secretomes of PDAC cells as well as PDAC tissues, were used for the SELEX process. White et al. described an in-vitro positive/negative selection strategy to identify a cyclophilin B RNA-aptamer (M9–5) that detects structural differences between the secretome of pancreatic cancer and non-cancerous cells. M9–5 has the ability to discriminate sera from PDAC-patients and healthy volunteers with high specificity and sensitivity , 45. AnoAptamer-based targeted imaging is one of the most promising molecular imaging technologies for PDAC diagnosis, accurate staging, and the monitoring of treatment response. Compared to conventional imaging protocols, aptamer-based targeted imaging employs tumor-specific, labeled aptamers to explore biological targets in living subjects. This method harbors the following advantages: i) early detection is much easier to achieve due to amplified imaging via aptamers targeting PDAC markers; ii) aptamers that specifically target PDAC biomarkers have the potential to differentiate between malignant and benign diseases; iii) more accurate imaging helps to evaluate precise staging of PDAC and improves surgical guidance for complete tumor resection and lymph node clearance; iv) aptamers support the detection of biological characteristics of PDAC patients for clinical decision making and prognostic prediction, which can be evaluated without biopsies .111–125 peptide antigen. Signals of the Cy3-conjugated aptamer were specifically localized on the surface of cancer cells from seven different entities, including pancreatic cancer. These in-vitro results suggest that Ap52 may have potential for future molecular imaging [A few aptamer-based, PDAC-targeted imaging approaches have been evaluated in-vitro and in-vivo Table , Fig. 1. imaging . In a xe imaging . TherefoAlthough there are no fluorophore-labeled aptamers available for clinical applications, human tumor tissue sections have been used to determine the performance of aptamer-based imaging in PDAC patient samples. Targeting PDAC cells with Cy-3-coupled P19/P1 aptamers was employed as a diagnostic tool on archival human pancreatic duodenectomy tissue sections. Scoring patterns from 72 patients revealed a positive correlation between high fluorescence signal intensity and significantly increased mortality . Thus, a3O4 was synthesized. This molecule can specifically bind to pancreatic cancer cells in vitro and hereby reduce MRI T2-weighted imaging signal intensity [Besides fluorescence imaging, magnetic resonance imaging (MRI) is another option for PDAC-targeted molecular imaging utilizing metal-oxide-labeled aptamers. Recently, a novel matrix metalloproteinase 14 (MMP14)-targeted aptamer (M17) conjugated with polyethylene glycol-Fentensity . As a reIn the past two decades several antibody-based, molecular-targeted therapies were implemented for the treatment of different hematologic and solid malignancies. Unfortunately, no antibody-based targeted therapy has yet been successful in improving the prognosis of PDAC patients . SimilarSeveral aptamers were identified for possible treatment strategies in PDAC Table . Kim andOligonucleotides, such as microRNAs (miRNAs), miRNA inhibitors (anti-miRs), antisense oligonucleotides (ASOs), and short interfering RNAs (siRNAs), have been shown to potently silence the expression of their target genes. However, one of the most significant obstacles for oligonucleotide-based therapeutic strategies is the lack of specific delivery to the tumor . AptamerNucleoside analogs are a class of drugs that are of special interest for aptamer-based targeted treatment. Due to the similar structure of nucleoside analogs and natural nucleosides, they can be incorporated into aptamers rather easily. Partha Ray et al. utilized a nuclease resistant RNA-aptamer that binds EGFR on pancreatic cancer cells and is subsequently internalized in order to deliver gemcitabine-containing polymers into EGFR-expressing cells, which inhibited cell proliferation in vitro . Dua et The original AS1411 aptamer mentioned above is a 26-nucleotide guanosine-rich DNA-aptamer with high affinity and specificity to nucleolin, a cell-surface receptor overexpressed in cancer cells. Because AS1411 has not only proven anti-cancer effects in models of various cancer entities, but is also an enhancer of cellular uptake, it is frequently used for the generation of aptamer-based drug delivery systems . AccordiCD71 (transferrin receptor) and EGFR are also commonly used targets in aptamer-guided therapy of PDAC. Several groups loaded doxorubicin, monomethyl auristatin E (MMAE), or monomethyl auristatin F (MMAF) to CD71- or EGFR-targeted aptamers, respectively , 70. The165, was approved by the US Food and Drug Administration (FDA) as an anti-angiogenic treatment for neovascular (wet), age-related, macular degeneration (AMD) in 2004, an increasing number of aptamers have successfully entered clinical trials. Until now, three aptamers have entered phase III. The protagonists are Pegnivacogin (RB006), a direct factor IXa inhibitor; E10030, an anti-platelet-derived growth factor (anti-PDGF-B) aptamer; and Zimura, a complement factor C5-inhibitor [Since pegaptanib, an RNA-aptamer directed against VEGFnhibitor .So far, NOX-A12 is the only aptamer undergoing a clinical trial for pancreatic cancer . NOX-A12In order to improve the therapeutic outcome of clinical PDAC treatment, it seems inevitable to design novel strategies that combine different treatment modalities aiming at achieving synergism . HoweverThree strategies for the combination of aptamers and ionizing radiation can be found in the literature. The first strategy uses aptamers, which exhibit radiosensitizing properties per se without any further conjugation since binding to their designated targets interferes with radioresistance signaling Fig. a. This wThe second radiosensitizing strategy utilizes aptamers as vehicles for radiosensitizing compounds Fig. b. This gleft panel). This, in turn, leads to an increase in the levels of free radicals in tumor cells and enforced DNA damage. One example of aptamers in this group is the already mentioned AS1411 aptamer against cell surface nucleolin. A gold nanocluster conjugate of the AS1411 aptamer enhanced the efficacy of radiation therapy in breast tumor-bearing mice. Importantly, the authors reported a specific enrichment of the radiosensitizing gold nanoclusters in the breast tumors and a significant increase in the mean survival times of the animals [Aptamers of this group enforce the biological effects of radiation by physical dose enhancement via photoelectrons, Auger electrons, and secondary electrons that are generated from irradiated metal particles  . This treatment strategy was successfully reported in an approach with targeted administration of DNA-PK silencing siRNA by aptamer-siRNA chimeras against prostate-specific membrane antigen (PSMA) in experimental models of prostate cancer. Upon aptamer treatment, an increase in radiation-induced cytotoxicity (based on specific DNA-PK knockdown) was achieved in vitro in a PSMA-positive prostate cancer cell line. The subsequent in-vivo evaluation with xenotransplants showed a PSMA-specific, significant delay in tumor growth upon the combination of aptamer treatment and radiotherapy with 6 Gy, which was not observed without irradiation or in non-PSMA expressing tumors, respectively [Aptamers in this category enhance the biological effectiveness of radiotherapy by their conjugation to substances that interfere with the DNA damage response  cells by combining tumor-specific radical-amplifying gold nanoclusters and tumor-specific inhibition of STAT3 signaling .right panel). As already described, pyrimidine analogs, such as 5-FU and gemcitabine, can directly be integrated into the molecular structure of aptamers thereby optimizing therapeutic drug delivery. Analogously, aptamers were also modified for the delivery of therapeutic radionuclides activate anti-tumor immune responses Fig. d. In a bAlthough aptamers in combination with radiation therapy are rarely explored in pancreatic cancer, the strategies we summarized above provide us promising approaches to sensitize radiotherapy in several cancers. Considering the high degree of radioresistance in PDAC, these aptamer-based radiosensitizing treatments supply us with novel ideas to decrease its radioresistance and maybe improve the prognosis in the future, especially regarding the emerging new technical possibilities of image-guided irradiation (e.g. MR-Linac).As described in this review, aptamers are very useful tools and have several applications in the diagnosis and therapy of pancreatic cancer. Unique features including ease of synthesis, high sensitivity and specificity and stability in different conditions, render aptasensors an interesting choice for the detection of biomarkers, CTCs, and CSCs. Additionally, aptamer-based biosensors represent ideal noninvasive devices for PDAC diagnosis even in early tumor stages. With help of the blind SELEX method, aptamers are able to identify novel biomarkers from cancer cells, secretomes, membranes or tissues, which can stimulate novel ideas for screening attempts and new hypotheses for mechanistic studies. Aptamer-based in-vivo imaging is an excellent way to obtain comprehensive images and molecular information without invasive biopsies. Data from aptamer-enhanced technologies will support clinical decision making and enhance the quality of precise prognostic prediction. Importantly, as aptamers can exert several ways of action, they are attractive tools for targeted therapy of PDAC, alone or in combination with standard approaches, such as radiotherapy. Aptamers can work as inhibitors and drug carriers. Their excellent performance in targeted inhibition renders them competitive alternatives to antibodies. Diverse aptamer-based drug delivery systems with incorporated nucleoside analogs, oligonucleotides, and other drugs allow aptamers to guide targeted chemotherapy or RNA interference into PDAC cells.However, the application of aptamers has some limitations. Significant problems include degradation in blood and high renal excretion , althougWith the development and advancement of aptamers, these molecules are becoming an attractive platform for translational applications. To make the best use of aptamers, future directions should focus on aptamer-based biosensors and aptamer-based, targeted drug-delivery systems – particularly systems for nucleoside analogue and oligonucleotide delivery for combined modality treatment approaches.


There is currently a requirement for single-sex litters for many applications, including agriculture, pest control, and reducing animal culling in line with the 3Rs principles: Reduction, Replacement, and Refinement. The advent of CRISPR/Cas9 genome editing presents a new opportunity with which to potentially generate all-female or all-male litters. We review some of the historical nongenetic strategies employed to generate single-sex litters and investigate how genetic and genome editing techniques are currently being used to produce all-male or all-female progeny. Lastly, we speculate on future technologies for generating single-sex litters and the possible associated challenges. Animal models remain indispensable experimental reagents for understanding fundamental biology and translational research. Despite this utility, there is an ongoing issue with the production of animals that are surplus to requirement. For example, in 2017, in Great Britain alone, over 1.8 million laboratory animals were culled without ever being used for a scientific procedure [The requirements for all-female or all-male litters is not limited to laboratory models . For exaSRY/Sry (Sex-determining region Y), which is expressed in Sertoli cell precursors [SRY/Sry mode of sex determination is not the primary method of sex determination for all mammals. For example, the platypus, a prototherian mammal, does not have an SRY gene [The production of all-female or all-male litters by genetic methods is feasible because in some species, males and females differ in their sex-chromosome complement . Eutheriecursors –17. It iSRY gene ,19.doublesex that undergoes sex-specific alternative splicing to regulate sex determination in many insect species, including Drosophila melanogaster, reviewed in [Conversely, in many bird species, including chickens, females are heterogametic and carry a single Z and a single W chromosome. Males are homogametic and carry 2 Z chromosomes. Avian sex determination is controlled by the dosage of a Z-linked gene called DMRT1 . Femaleiewed in .In this Review, we investigate some of the current requirements for single-sex litters in research and in other industries such as agriculture and pest control. We describe some of the historical methods for sexing-sorting and advantages and disadvantages associated with them. We then discuss current methods performed to generate single-sex progeny, including by genome editing methods such as CRISPR/Cas9 –26. FinaHistorically, sex selection is performed by investigating sex-specific biological differences. For example, male and female chick embryo allantoic fluid contains differential levels of estrone sulfate . HoweverGiven the risk of embryonic nonviability and pain perception associated with invasive procedures for sexing embryos, one superior method to selecting offspring sex is by separation of X- or Y-carrying sperm. Prior selectivity of sex-specific gametes for in vitro fertilisation (IVF) or artificial insemination (AI) ensures that the offspring sex is predictable, which may be more economically viable and ethically justifiable than invasive sexing procedures.Many techniques have been previously attempted to isolate X- and Y-sperm, including fluorescence in situ hybridisation (FISH) and swim separation . The mosFlow cytometry is associated with large economic and time costs in sperm sorting and postsort procedures (reviewed in ), leadinAnother technique also utilising cell-surface markers for separating X- and Y-sperm, used in bulls, is ‘WholeMom’ . In thisIn summary, current approaches rely on the differential DNA content or surface markers of X- and Y-carrying sperm in order to physically separate the sperm. These strategies rely on IVF of sex-sorted sperm to skew offspring sex ratios. Although these and other methods not described in detail in this Review . To selBombyx mori (Mulberry silkworm). Male silkworms are desirable over females because they produce higher-quality silk [doublesex minigene from Pectinophora gossypiella  and inserted the transgene into the B. mori genome [Pgdsx doublesex undergoes sex-specific alternative splicing; therefore, in B. mori, tTAV expression was specific to females. The female-specific tTAV protein accumulation induced female-specific lethality, resulting in male-only cocoons surviving. Moreover, the female-specific lethality could be largely repressed by the addition of dietary tetracycline [doublesex-regulated lethality constructs integrated into other pest insect genomes—for example, the olive fly and Mediterranean fruit fly—did not have the same lethality effect [A refinement of SIT is called ‘Release of Insects carrying a Dominant Lethal’ . RIDL iity silk , but RIDi genome . Endogenacycline , allowiny effect ,86.D. melanogaster [transformer or doublesex genes. Male and female flies have normal viability until the dietary addition of either puromycin or geneticin, which selects for males or females, respectively, producing progeny of 100% the required sex.More recently, Kandul and colleagues generated an antibiotic-resistance–based sex-selection transgene system in nogaster . Two druAlthough the use of fluorescence-transgene–based sexing systems were inefficient in insect species, in chickens, they are currently the most promising genetic approach for chick sexing prior to hatching. In Australia, the Commonwealth Scientific and Industrial Research Organisation (CSIRO) is championing a chick-marker approach whereby a fluorescent marker is integrated onto the male-determining Z chromosome so that male and female chicks can be segregated prior to hatching .Anopheles mosquito model [Anopheles males are heterogametic, XY, whilst females are XX. An I-PpoI  cassette was genetically engineered onto the Y chromosome. The I-PpoI endonuclease selectively targeted the X chromosome, resulting in endonuclease-driven shredding of the X [doublesex intron 4–exon 5 boundary, driving I-PpoI to induce X-shredding [A superior system of generating single-sex litters in the laboratory and agriculture would be to produce a single type of sex-chromosome–carrying gamete, i.e., only X- or only Y-carrying sperm, by selective destruction of the unrequired sperm. Evidence for the first genetic methods to skew offspring sex ratios by destruction of the X-carrying sperm was demonstrated in the to model . Anophelof the X ,90. The of the X –92. Usinof the X . Most rehredding .Drosophila [RpS6  and RpS5a  generated a male-biased offspring sex ratio skew from 93.8% to 56.6%, respectively. This study highlighted that although the X-shredding strategy was applicable to other species outside mosquitos, X-meddling proved to be a more efficient method of producing sex-biased progeny and may be more greatly applicable to other species.Faluso and colleagues expanded on the Galizi and colleagues ,93 studiosophila . They prAnother CRISPR/Cas9-based method for generating single-sex litters is a bicomponent system. A bicomponent CRISPR/Cas9 system refers to the genetic isolation of Cas9- and sgRNA-encoding transgenes. The isolated Cas9 and sgRNA transgenes are coinherited independently of each other from either parent. Integration of either the Cas9 or sgRNA transgene onto a sex chromosome of a heterogametic parent allows for sex-specific inheritance of the transgene. Monoinheritance of either the Cas9 or sgRNA transgene in isolation is predicted to have no mutational effect and therefore is advantageous because monoallelic stocks can be bred normally. Furthermore, it is known that constitutive expression of transgenic Cas9 is not detrimental to mice –98. ConvB. mori [Bmtra2 (B. mori transformer 2). Coinheritance of the W-Cas9 and sgRNA transgenes in daughters resulted in Bmtra2 CRISPR/Cas9-induced mutations, 50% of the progeny did not hatch, and the surviving progeny were 100% male. This study was the first to highlight that sex-specific coinheritance of CRISPR/Cas9 components targeting an essential gene produces single-sex litters. Developments in generating single-sex offspring by genetic methods has the potential to be extremely advantageous for the silkworm industry.The first implementation of a CRISPR/Cas9 bicomponent system to breed all-male offspring was in B. mori . SimilarB. mori . Second Bmtra2 mutations and female-specific lethality. The same principle is relevant to eutherian mammals; integration of a Cas9 transgene onto the Y chromosome would ensure unique inheritance by sons. Coinheritance of a Y-linked CRISPR/Cas9 component transgene and autosome-linked CRISPR-Cas9 component will induce mutations in the target gene uniquely in sons. If the target gene is an essential viability gene, the sons will be embryonic lethal.In the Zhang and colleagues silkworm study, the aim was to produce all-male progeny. However, laboratories or agricultural applications may require all-female litters; for example, female rodents to study female-specific biology or in producing layer hens . In the SRY/Sry approximately 148–166 million years ago [However, the Y chromosome has been extremely challenging to genetically modify because of its highly heterochromatic and repetitive nature. The modern eutherian X and Y chromosomes diverged from a pair of ancestral autosomes  followinears ago . Recombiears ago . The Y cears ago . The cloears ago ,103,104 ears ago . The losSry promoter, was randomly integrated into the genome by zygotic microinjection [Sry-promoter–driven transgenic alleles, however, is that mouse Sry expression is tightly regulated in the gonad, occurring between embryonic day (E) 10.5 and E12.5 [Given the complexity associated with Y-gene targeting, the first study generating a targeted mammalian Y-gene reporter was not published until 2013, using transcription activator-like effector nucleases . Previonjection . The maind E12.5 –16. TherSry-promoter–driven expression is restricted, a preferable choice would be a promoter driving a Y-gene with ubiquitous expression. There are multiple Y-linked genes ubiquitously expressed in the mouse, including Uty  [Eif2s3y  [Ddx3y  [Kdm5d (Lysine demethylase 5D) [Uty is expressed during embryonic development and also in embryonic stem cells (ESCs) [Uty-eGFP reporter ESC lines [Uty and Ddx3y, thereby opening up new possibilities of Y-chromosome knock-in targets.Given that -linked) , Eif2s3y-linked) , Ddx3y (-linked) , and Kdmlase 5D) . Of thess (ESCs) . FurtherSC lines . MoreoveSC lines . The traUty locus to integrate a constitutively expressed sgRNA transgene into the second intron [Atp5b , Casp8 (Caspase 8), and Cdc20 (Cell division cycle protein 20) and an autosomal constitutively expressing Cas9 transgene resulted in knock-out of the target loci and significant male-biased offspring sex ratio [Uty expression was affected in these sgRNA-transgenic males was not addressed.In 2019, the first mammalian CRISPR/Cas9 bicomponent system was described, with the intention of generating all-female litters by CRISPR/Cas9-induced knock-out of essential genes in male embryos. Yosef and colleagues utilised the ubiquitous Y-linked d intron , which wex ratio . WhetherRosa26)-Cas9 hemizygous transgenic mothers, maternally loaded Cas9 was sufficient to induce mutations even in non-transgenic offspring [Bicomponent systems are advantageous because mono-transgenic stocks can be maintained as separate lines and bred when necessary. The human control of using original stocks maintained independently ensures that genetic mutational resistance at the target loci is unlikely to occur. Furthermore, if mutational resistance did arise in any offspring, the individuals can be removed from the population without any negative impact on the breeding stocks. Refining the technologies further, it may be possible to generate transgenic laboratory models wherein the transgenes that induce the embryonic lethality effect are inherited by maternal deposition of mRNAs. In an analysis of embryos derived from Gt(ROSA)26Sor . Interestingly, homozygous targeted females were not sex reversed but instead showed an intersex phenotype and were infertile [In 2018, Kyrou and colleagues generated a gene-drive system in mosquitos, targeting the sex gene . In thisnfertile . This stTyrosinase (Tyr) gene, which generates white-coated mice upon homozygous knock-out. An sgRNA transgene targeting Tyr and also encoding an mCherry reporter was inserted into the Tyr locus to produce a Tyr-heterozygous knock-out and hemizygous transgenic mouse line. When bred with Cas9-expressing mice, functional gene-drive systems would transmit to offspring at above-mendelian frequency. A successful gene drive should produce Tyr loss-of-function white mice that also express mCherry. In this approach, gene-drive success varied from 0% to 72%. Often, the mice were white-coated but did not express mCherry, suggesting that transmission of the sgRNA and Cas9 transgenes produced mutations at Tyr but without copying the sgRNA/mCherry transgene [Tyr was likely nonhomologous end-joining (NHEJ), consistent with previous reports that NHEJ is the dominant mode of repair over homology-directed repair [To examine whether the synthetic suppressive drive systems could also be applied to mammals, Grunwald and colleagues performed the first proof-of-principle gene-drive system in mice . To asseransgene . The repd repair ,122. Thed repair , mammaliDrosophila [Drosophila CRISPR/Cas9 transgene expression for X-meddling or X-shredding is still open; however, great strides have been made in determining possible transgene integration sites [Genetic or genome editing systems have the potential to effectively generate single-sex litters; however, they currently have some disadvantages. The superior method of generating single-sex litters is by selective destruction of the nonrequired sperm. One method to selectively destroy X-carrying sperm is by X-shredding or X-meddling. Although this technique was shown to be highly efficient in mosquitos ,93, it wosophila . An impoon sites .Although CRISPR/Cas9 bicomponent systems are advantageous in that the mono-transgenic stocks can be easily maintained independently, current bicomponent systems are not 100% efficient. In the Yosef and colleagues study , the sexOne possible disadvantage of a released gene-drive genetic system is through mutational resistance arising at the sgRNA target site. If a nucleotide variant arises at the sgRNA target or the neighbouring protospacer-adjacent motif (PAM), then the CRISPR/Cas9 system becomes immediately dysfunctional. Mutational resistance to an embryonic-lethal gene drive could potentially spread efficiently through the population because of conferring a fitness advantage. Indeed, evidence for rapidly arising resistance alleles has been shown recently in flies  and mosqA second disadvantage associated with CRISPR/Cas9 gene drive is the risk that the above-mendelian frequency of inheritance spreads so rapidly through the population that the original wild-type allele is completely lost. The complete loss of the wild-type allele may cause apprehension against gene drive. Adaptations to gene-drive methods have been developed in order to prevent the uncontrolled spread of synthetic gene drives, called ‘self-exhausting’ gene drives, for example, the killer-rescue  or daisyOverall, there are many challenges still associated with producing transgenic mouse lines in order to produce single-sex litters. However, with careful consideration of which genes are targeted to induce embryonic lethality or sterility, bicomponent CRISPR/Cas9 methods could be widely employed to skew sex ratios. For example, targeting genes that are essential in postimplantation development may not result in a complete loss of the unrequired sex. Instead, an alternative approach may be to target essential housekeeping genes with roles in preimplantation development at embryonic genome activation. Embryonic lethality can then be induced very early, before the onset of organogenesis. Inducing embryonic lethality at preimplantation ensures firstly that embryos are nonviable prior to the onset of pain perception. Secondly, it would be interesting to determine whether embryo loss due to nonviability prior to implantation may allow extra viable embryos to implant, thereby compensating the litter size.Rosa26-Cas9 may circumvent issues with genetically modified offspring for agricultural produce. Moreover, it may be that some consumers consider the use of transgenic animals in agriculture ethically preferable to the widespread culling of the unrequired sex. Indeed, there are some examples of genetically modified produce currently in the food industry, for example, a modified salmon species with increased growth rate [Even if all of the necessary optimisations are made and the technology for generating single-sex litters is consistent, there may be some apprehension regarding using genetically modified animal produce in agricultural industries. Utilising maternally loaded mRNAs such as the earlier described wth rate , althoug