Climate change has caused frequent extreme low-temperature events to threaten global food security. Spring low-temperature stress is one of the major limiting factors for high and stable yields of wheat. We used two wheat varieties differing in spring cold-sensitivity (cold-tolerant variety Yannong 19 and cold-sensitive variety Xinmai 26) to examine the effects of low-temperature stress during the anther differentiation period on wheat photosynthetic performance and spike-setting characteristics. Low-temperature stress was simulated in a climate box at −2 °C, 0 °C or 2 °C (night) and 15 °C (day) for 24 h, 48 h or 72 h. With the extension of the treatment time and the decrease of temperature, the photosynthetic rate, stomatal conductance and transpiration rate of wheat leaves gradually decreased. All treatments except −2 °C for 72 h recovered slowly within 7–15 days after treatment. Low-temperature stress greatly reduced grains per spikelet, 1000-grain weight and yield per plant. By analyzing the spikelets in different stalk locations , we found that the number of upper spikelets was significantly less than lower and middle spikelets after low-temperature stress. The sterile grain of upper spikelets  can reach 100% at −2 °C for 48 h and 72 h, and the yield loss rate was 90.52% at 2 °C for 24 h, which was much higher than for the lower spikelets (60.73%) and middle spikelets (50.94%). Overall, these findings suggest that low-temperature stress during the anther differentiation period alters the photosynthetic activity involved in the accumulation of dry matter in wheat, which leads to delaying young spike growth, especially for upper spikelets, and ultimately in a decrease in yield. The worre years . In the re years ,15 have re years .Low-temperature stress in spring means that the temperature in the season of rebirth and growth  rises faster, and in the late spring  the temperature is lower than normal. When the development of winter wheat enters the anther differentiation period, the young ears’ resistance to frost will drop sharply . Once itA large number of studies have shown that low-temperature stress at the booting stage can significantly reduce the photosynthetic rate of major functional leaves, such as the flag leaf and the second and third fully developed leaf ,19, causPhotosynthesis is the source of plant material metabolism and energy transformation . It is sIn the present study, controlled phytotron experiments were conducted with different cold-sensitive winter wheat cultivars under different low-temperature levels and durations during the anther differentiation period. Our primary objectives were to (1) analyze the effects of low-temperature level and duration during the anther differentiation period on leaf photosynthetic properties, and (2) quantify the effects of low-temperature level and duration on spike-setting characteristics, especially wheat spikelets at different positions.Test materials: the cultivars Yannong 19 (cold-tolerant variety expressed by YN19) and Xinmai 26 (cold-sensitive variety expressed by XM26), as the main varieties cultivated in the Huang-huai wheat area , were se−1; available nitrogen 112.2 mg kg−1; available phosphorus 23.0 mg kg−1; available potassium 161.6 mg kg−1) was loaded into the test pots , which were then buried in the test field. Every pot was filled with 10 kg of soil; 11.52 g of compound fertilizer (N:P:K = 15:15:15) was applied to each pot before sowing, and 3 g of pure nitrogen was applied to each pot during the wheat jointing stage. 14 seeds were planted per pot, and 7 seedlings were reserved at the wheat trefoil stage.Test treatment: 0~20 cm tillage layer soil in the field . When the young ears reached the anther differentiation period , all the pots except the control treatments (expressed by CK) were moved to the RXZ smart climate box  with humidity 70% and light 17,000 LX during the day and 0 LX at night for treatment .Determination of photosynthetic parameters: the second fully developed leaf from the growing point was selected, and the photosynthetic indexes were measured by using a LI-6400  portable photosynthesis system; relative chlorophyll content (SPAD) was measured with a SPAD-502 .2 concentration (Ci).Photosynthetic indexes measured were: net photosynthetic rate (Pn); transpiration rate (Tr); stomatal conductance (Gs); and intercellular COThe wheat was harvested at maturity, and the yield factor was calculated by recording the number of spikes per plant, number of grains per spikelet, 1000-grain weigh and yield per plant.After low-temperature treatment, wheat leaves were selected to measure four photosynthetic indexes. Each treatment was repeated 3 times. At maturity, 3 plants were randomly selected from each treatment in order to assess the number of spikes per plant, grains per spike, grain yield per plant, and yield for the upper, middle and lower regions (6–7 spikelets per region). All pots were used for the 1000-grain weight.Data were subjected to analysis of variance , and statistical divergence among treatments was determined using Duncan’s new multiple range test. Correlation tests were calculated using KMO (Kaiser–Meyer–Olkin). Statistical analyses were conducted using SPSS statistical software .The three-factor variance analysis of varieties, treatment temperature and time of various photosynthetic parameters showed that the photosynthetic parameters and the three-factor interaction were extremely significant 3 days after treatment, and then the correlation gradually weakens. The correlation was significant again at 15 days after treatment. .Under the same low-temperature level, but with the extension of the low-temperature duration, the leaf Tr of the two wheat varieties showed a decreasing trend c,d, whilThe Gs of the two wheat cultivars under low-temperature treatment was basically consistent with the Tr e,f. With2·mol−1. With the decrease of temperature and the extension of treatment time, Ci had an increasing trend (In the control treatment, the Ci of the two varieties was not much different—both were about 200 μmolCOng trend g,h.SPAD of 72 h treatment leaves was significantly lower than others up to 15 days after treatment . Except The response to the temperature treatments varied depending on the position of the wheat spikes. Variety, treatment temperature and treatment time all had a significant effect on the upper-spikelet grain number.The three factors had extremely significant effects on the 1000-grain weight of the wheat. The yield of the upper spikelets was greatly reduced in both varieties and at each treatment time. .Low-temperature stress during the anther differentiation period affected the number of grains per spike. Some spikelets and florets were damaged, resulting in a delay or even cessation of normal differentiation and development. Ultimately, yield was reduced. With the decrease of the treatment temperature and the extension of the stress time, the number of grains per spike decreased, and grain-setting loss rate increased—the difference between the treatments was significant.With the decrease of the treatment temperature and the increase of the treatment time, the number of grains showed a decreasing trend from the middle spikelets to the upper and the lower spikelets. The upper spikelet grain-setting loss rate was the largest.  CultivarLow-temperature stress during the anther differentiation period has a great influence on wheat yield per plant, which is positively correlated with the intensity of treatment. The rate of spikelet setting and 1000-grain weight were decreased.Under low-temperature stress, the yield-reduction range at different positions of the wheat spike varied. The yield decrease of upper spikelets was much higher than lower and middle spikelets. The degradation of upper spikelets was the main cause of yield reduction .The Pn of leaves after low-temperature treatment has a significant positive correlation with the grains per spike and the yield. The correlation weakens with the passage of time after treatment.The correlation between Pn and 1000-grain weight of XM26 has an extremely significant correlation after treatment, which is different from YN19 .Photosynthesis is one of the physiological processes most sensitive to low temperature. A number of studies have implicated the effects of low temperature stress on photosynthesis of wheat leaves ,30. HoweThe decrease of Pn caused by low temperature is accompanied by the decrease of Gs . SimilarThis study showed that low-temperature stress during the anther differentiation period can reduce the number of spikes per plant and the number of grains per spike. The decrease in grain number per spike is due to the changes in photosynthesis, which affects dry-matter accumulation . Low-temLow-temperature stress during the anther differentiation period not only reduced the number of grains per spike but also 1000-grain weight. Xiao et al.  analyzedIn conclusion, low-temperature stress affects the relationship between photosynthetic performance and spike-setting characteristics which means making the source–sink relationship of wheat unbalanced , finallyUnder the conditions of climate warming, cultivating and selecting varieties resistant to low-temperature stress in spring is an important way for wheat to resist spring low temperature and to provide a stable yield. The results of our experiment showed that low-temperature stress during anther differentiation has serious effects on photosynthesis, spike-setting characteristics and yield of wheat. In the breeding and identification of varieties, photosynthetic indicators, number of grains per spike, etc., can be used to evaluate the frost tolerance of wheat reference indicators in order to focus on breeding low temperature stress resistant variety. The results also preliminarily showed that the low-temperature stress during anther differentiation especially effects the upper spikelets of wheat—the difference in yield among varieties was mainly due to the difference in seed setting of upper spikelets. Low-temperature stress during anther differentiation affects the grain-setting characteristics of wheat mainly through affecting the photosynthetic characteristics of leaves.In future work, the physiological and molecular mechanisms affecting spike development and grain establishment in wheat will be explored, thus providing support for disaster prevention and mitigation of wheat production.


Recent case reports and studies on treating COVID-19 in patients with chronic sarcoidosis describe different treatment modalities ranging from glucocorticoids to biologic medications. This review article summarizes seven case series and reports totaling 46 patients. While one case report suggested that sarcoidosis medications such as glucocorticoids may lengthen the COVID-19 disease course, another study with a larger registry suggests they do not. More studies are needed to elucidate an improvement in outcomes. It is possible that addition of TNF-alpha inhibitors at COVID-19 diagnosis decreases hospitalization rate. Overall, it is difficult to make firm conclusions regarding treatment given the heterogeneity of treatment modalities in the current literature. Our summarized findings are outlined with the opinions of sarcoidosis, pulmonary, and infectious disease experts in a flow chart that provides clinicians with our proposed management algorithm for sarcoidosis patients who develop COVID-19. We emphasize a need for exchange of information regarding management of COVID-19 in the setting of sarcoidosis to further improve treatment in this vulnerable population of patients. Sarcoidosis is a multi-system inflammatory disease of unknown etiology. Most commonly, it presents as a pulmonary disease with bilateral hilar adenopathy, pulmonary reticular opacities, and non-caseating granulomas. However, it may present with cutaneous, ophthalmologic, musculoskeletal, cardiovascular, or CNS lesions. Though the archetypal patient in the United States is a middle-aged black female patient with hilar adenopathy, this characterization poorly captures the variety of findings in practice. For example, men are diagnosed earlier than women and Scandinavians are another common ethnic population to develop the disease . This isSarcoidosis is a diagnosis of exclusion with corresponding clinical and radiographic findings . Most paOver the past year, a COVID-19 has swept the globe and presents new challenges to the management of pulmonary disease. Importantly, comorbidities indicated in COVID-19 deaths, hypertension, diabetes, cardiovascular disease, and respiratory system disease, are also comorbidities in patients with sarcoidosis , 6. ReceUntil June 6th, 2021, the following search criteria with Boolean logic was utilized on the PubMed, Scopus, and Cochrane Library databases: (sarcoidosis) AND (Covid-19 OR SARS-CoV-2 OR Coronavirus disease 2019). Search results from the Scopus database were further limited to open-access articles in the subject area of Medicine and in the final publication stage. A total of 30 publications were identified and sorted by relevance. Inclusion criteria were case report or case series of patients with chronic sarcoidosis. Exclusion criteria were papers without mention of sarcoidosis baseline treatment, management of COVID-19, or COVID-19 outcomes . Seven pThe seven publications reviewed had shared commonalities, which are reported in this paper, as well as differences which will be highlighted here. There is inconsistent data provided on existing sarcoidosis treatment and respective changes made to these medications. Many publications provide dosage and dosing schedule, while some only provide the type of medication. Case reports provided information such as the day of discharge relative to initiation of care while case series noted deaths, suspected cause of death, and other eventful outcomes such as bacterial infection. Further, there is a lack of systematic prospective studies and no control groups. There may be biases in the reporting of COVID-19 and its complications by patients and their primary physicians. Thus, data retrieved from tertiary centers may be biased. This heterogeneity in data reporting underscores the difficulty in drawing methodological conclusions from these publications and highlights the need for greater research on this subject matter. We recognize this as a limitation to our review and encourage further sharing of COVID-19 data in sarcoidosis patients.Our literature review yielded case studies, case series, and guidelines for treating patients with COVID-19 superimposed on sarcoidosis. We have summarized the findings of seven case series and reports totaling 46 patients in p = 0.003) (An examination of 7,337 COVID-19 patients from a hospital system in New York City found 37 people with concomitant sarcoidosis. Of this population, 14 had moderate to severely impaired pulmonary function with nine succumbing to COVID-19 whereas only three of the 23 without these physiologic deficits succumbed to COVID-19. This data shows those diagnosed with COVID-19 with decreased pulmonary reserve increases the risk of adverse outcomes, measured by increased rates of intubation and hospital mortality (A series of correspondences published in the Annals of Rheumatic Diseases discussed the role of corticosteroid modification in the treatment of patients with sarcoidosis who developed COVID-19 and later highlighted potential protective qualities of TNF-alpha antagonists. The first correspondence by Györfi et al. reported a case of COVID-19 relapse in a patient with Löfgren syndrome diagnosed in 2019 after apparent resolution of COVID-19 and a negative COVID-19 PCR test 14-days after exposure . 5 days Among non-sarcoidosis patients, there is evidence that long-term glucocorticoid treatment ≥10 mg/d may play a role in worsening COVID-19 outcomes among patients with systemic lupus erythematosus . In a stJeny et al. also discussed possible protective qualities that prior TNF-alpha treatment may have had on sarcoidosis patients with a new COVID-19 diagnosis. Six patients with prior TNF-alpha antagonist treatment had their treatment suspended to mitigate further immunosuppression. Of the six, one patient died of hypercapnia secondary to chronic obstructive pulmonary disease and obesity and the rest were not admitted to the ICU during their disease course. Consistent with previously published data, the severest forms of COVID-19 in Jeny et al.'s data were not associated with prior TNF-alpha antagonist treatment –19. In aPadala et al. presents a unique case of a pulmonary and cardiac sarcoidosis patient with a varied immunosuppressive home regimen prior to COVID-19 diagnosis: 40 mg biweekly subcutaneous adalimumab, 40 mg daily prednisone, and 20 mg weekly methotrexate . In ApriThere was debate on the usage of antimalarials in the treatment of COVID-19. Yates et al. described a case series which included the administration of doxycycline to four COVID-19 patients with varying medical history . A patieFor over a year, COVID-19 has presented a novel challenge for patients with chronic lung disease due to the respiratory disease pathology caused by SARS-CoV-2. We recognize that in the pandemic, the science for managing patients with sarcoidosis is rapidly evolving and that new findings may offer clearer management guidance. Here, we offer a suggestion in management  based onSK and TH wrote the bulk of the manuscript. MM worked with SK and TH to edit the contents of the paper as well as contributed several paragraphs of the manuscript. CA provided pulmonary disease expertise as well edited the contents of the manuscript. RN provided infectious disease expertise as well as edited the contents of the manuscript. NS provided expertise in sarcoidosis as well as edited the contents of the manuscript. All authors contributed to the article and approved the submitted version.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.


E gene, these 40 samples were re-isolated and subsequently re-analysed using the three kits, which detected the virus in 22, 23 and 12 cases, respectively. The results point to a divergence not only between antigen and RT-qPCR tests, but also within the “gold standard” RT-qPCR testing. Performance analysis of the diagnostic antigen test showed the positive predictive value (PPV) to be 100% and negative predictive value (NPV) to be 98.10%, indicating that 1.90% of individuals with a negative result were, in fact, positive. If these data are extrapolated to the national level, where the mean daily number of antigen tests was 250,000 in April 2021, it points to over 4700 people per day being misinterpreted and posing a risk of virus shedding. While mean Ct values of the samples that were both antigen and RT-qPCR positive were about 20 , mean Ct values of the samples that were antigen-negative but RT-qPCR-positive were about 30 . It confirms the advantage of antigen test in detecting the most infectious individuals with a higher viral load. However, the reporting of Ct values is still a matter of ongoing debates and should not be conducted without normalisation to standardised controls of known concentration. The global pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is having a tremendous impact on the global economy, health care systems and the lives of almost all people in the world. The Central European country of Slovakia reached one of the highest daily mortality rates per 100,000 inhabitants in the first 3 months of 2021, despite implementing strong prophylactic measures, lockdowns and repeated nationwide antigen testing. The present study reports a comparison of the performance of the Standard Q COVID-19 antigen test (SD Biosensor) with three commercial RT-qPCR kits  in the detection of infected individuals among employees of the Martin University Hospital in Slovakia. Health care providers, such as doctors and nurses, are classified as “critical infrastructure”, and there is no doubt about the huge impact that incorrect results could have on patients. Out of 1231 samples, 14 were evaluated as positive for SARS-CoV-2 antigen presence, and all of them were confirmed by RT-qPCR kit 1 and kit 2. As another 26 samples had a signal in the  Coronaviridae, genus Betacoronavirus and subgenus Sarbecoviruses  kit  uses Stabilized Amplification Technology-Nucleic Acid Testing . The RT-qPCR results were analysed using the instrument software (qPCRsoft 4.0) with a constant threshold line setting.All qRT-PCR analyses were performed on qTOWERThe IBM SPSS statistical program v.21 was used to calculate descriptive statistics. The paired T-test was used to compare the mean Ct values between diagnostic tests. A non-parametric Wilcoxon test for independent samples was used to compare Ct values between two diagnostic groups .The sensitivity of the rapid antigen test was calculated as true positive/ × 100, and the specificity was calculated as true negative/ × 100.We also calculated the performance of the antigen diagnostic test by the positive predictive value (PPV) and negative predictive values (NPV) as follows: PPV = true positive/ and NPV = true negative/.The results confirmed that the Standard Q COVID-19 Ag test (SD Biosensor) has an advantage in its ability to detect people with lower Ct values, who can be assumed to have higher viral load and thus pose the highest risk of virus transmission.The relatively high number of false-negative results from the antigen test increases the risk of silent virus shedding in the population. If used in mass testing, thousands of individuals could receive incorrect results and proceed to spread the virus, especially if tested at an early phase of viral infection.The antigen test is therefore time-limited, and its utilisation in blanket population screening should be re-considered given that the benefit vs. risk ratio is questionable.The sensitivity and specificity of an antigen test compared to the gold standard RT-qPCR testing is not accurate unless RT-qPCR sensitivity limits are included in the algorithm.Providing row Ct values is not sufficient. We proved that one sample can have variable Ct values if different assays are used. Moreover, the same Ct value of kits with different sensitivity represent a discrepant amount of virus copies (even in the thousands). Additionally, identical Ct detected by one kit can have different infectivity aspects depending on the disease phase.The algorithm for RT-PCR testing should also calculate actual health status, and if an early phase is suspected, retesting should be considered in the cases when Ct was detected above the threshold resulting in a negative result.Clear guidelines for pre-analytical, analytical and post-analytical steps, including chemistry quality, are needed to uniform the testing.Normalisation of Ct values by standard dilution curve, counting virus copies could allow inter-kit comparison between laboratories and epidemiological monitoring.To conclude the results of this study by summing up our experience and expectations, the main outcomes could be defined as:


Despite the tremendous efforts made in the past decades, severe side/toxic effects and poor bioavailability still represent the main challenges that hinder the clinical translation of drug molecules. This has turned the attention of investigators towards drug delivery vehicles that provide a localized and controlled drug delivery. Molecularly imprinted polymers (MIPs) as novel and versatile drug delivery vehicles have been widely studied in recent years due to the advantages of selective recognition, enhanced drug loading, sustained release, and robustness in harsh conditions. This review highlights the design and development of strategies undertaken for MIPs used as drug delivery vehicles involving different drug delivery mechanisms, such as rate-programmed, stimuli-responsive and active targeting, published during the course of the past five years. Certain categories of pharmaceuticals, such as peptides, gene-based drugs and small molecules with low solubility and/or lipophilicity, cannot be effectively delivered using conventional formulations, due to enzymatic degradation and/or insufficient absorption into systemic circulation because of molecular size and charge. A drug delivery system (DDS) refers to a device or a formulation that vehiculates a therapeutic agent into the body, with an adequate dosage and possibly in a targeted manner, at the desired rate and time to reduce adverse effects, improve patient compliance and extend the duration of pharmacological actions .The loading capacity of different DDSs can significantly affect treatment efficiency and/or dosing frequency in various ways, mainly by a change of the pharmacokinetic and pharmacodynamic profile ,3. In paNevertheless, a burst release of drug is frequently observed in conventional polymeric vehicles, thus resulting in potentially severe consequences for the patients such as toxicity issues and undesirable side effects caused by temporarily high-drug dosage . To dateMany approaches have been investigated to overcome the limitations existing in conventional polymer vehicles ,22,23. AThe affinity of polymers for template molecules during polymerization was first demonstrated by the seminal work of Polyakov in 1931 . HoweverMIPs are prepared in the presence of target molecules, called “templates”, used to generate specific and complementary binding sites within the polymer matrix. This allows MIPs to recognize the templates with antibody-like affinity and selectivity ,43. GeneIn the conventional preparation scheme of MIPs , the funHowever, the practical and commercial applications of MIPs prepared with free template molecules in solution have been limited by several issues, including template residues leftover in polymeric matrices, binding site heterogeneity and complex production processes . To addrThe use of molecular imprinting technology in DDSs is a novel and attractive field of research, which can generate efficient polymeric DDSs with the high specificity to recognize target biomolecules and the ability to introduce stimuli-responsiveness ,61,62. AAdditionally, owing to the high stability and robustness of MIPs in harsh conditions, MIPs have shown fascinating potential in drug delivery. Indeed, MIPs can be stored in a dry state at room temperature without losing their recognition properties for several years . FurtherThe considerations for the safety and biocompatibility of MIP-based DDS are extremely important. Acrylic and methacrylic monomers have been widely used in various MIP applications, and they can also be used to develop DDSs because of their biocompatibility . HoweverNorell, Andersson and Nicholls firstly reported in 1998 that MIPs can be potentially used as sustained drug delivery vehicles to release theophylline . They shThe MIP matrices rely on a high degree of cross-linking to maintain complementary cavities that act as drug reservoirs , but theTaking into account the above-mentioned aspects, as well as the exponential increase in literature examples of MIP-based DDSs during the course of the past five years, this review aims to highlight the design and development of strategies undertaken for MIPs used as drug delivery vehicles involving three main drug-delivery mechanisms: (i) rate-programmed drug delivery, where MIPs serve as excipients to control drug diffusion and/or enhance loading capacity for the system; (ii) stimuli-responsive drug delivery, in which some physical, chemical and/or biological factors can activate drug release from the system; (iii) active targeting drug delivery, where MIPs act as target delivery vehicles that provide spatial control of drug release to specific sites of the body. We will strive to provide comparisons with conventional and possibly commercial examples of polymeric DDSs, bearing in mind the administration routes, to identify the main challenges that need to be addressed in order to eventually actualize the commercial translation of MIP-based DDSs. Within the context of this release mechanism, MIPs are used as excipients to control drug release at a specific rate and/or time. Since Norell, Andersson and Nicholls first reported that MIPs could potentially serve as sustained drug delivery vehicles for theophylline release , it has MIP-based DDSs have been exploited for transdermal formulations due to their advantages of controlled release, enhanced drug loading and/or enantioselectivity, thereby reducing the required dosing frequency, decreasing side effects, and improving therapeutic effects . Nicotin−1, respectively, thus highlighting the effect of the presence of the selective cavities into the MIP. Skin penetration studies (on a porcine model) demonstrated that the permeation rate of the MIP formulation was close to the commercial nicotine patch Nicopatch® and followed zero-order kinetics up to 48 h. The MIP formulation exhibited a longer duration of action than the commercial nicotine patches Nicopatch® and Nicorette® ..91].In this system, the color change was achieved with an inverse opal architecture obtained by silicon nanoparticles (SiNPs). Owing to the presence of numerous imprinted cavities, the MIP-based contact lenses had ~2-fold higher loading capacity at pH 6.5 than the corresponding NIP-based contact lenses. In simulated lacrimal fluid (pH 8.0), timolol release was almost complete in 12 h, and no burst release was observed. It is worth mentioning that the lenses did not have any structural color in the central area of the pupil so as not to interfere with vision. Besides, the study found that the contact lenses were reusable for five cycles without a change in performance, thus indicating the potential of MIPs in the creation of a reusable and self-monitoring therapeutic contact lenses.−1 and 60 mg g−1, respectively) was lower than that of the optimal MIP formulation (100 mg g−1). However, the lipophilicity of polymyxin B impeded light transmittance: the MIP formulation transmitted less than 45% of light when loaded with more than 25 mg of polymyxin B. This limitation may impede the development of MIP-based contact lenses for loading lipophilic drugs, but the in vitro release study performed in saline solution showed that the MIPs sustained drug release for up to 14 days.The hydrophilicity of polymeric matrices is usually counterproductive for the delivery of drugs that are lipophilic and/or have high molecular weight. For instance, polymyxin B is an antimicrobial lipopeptide that can be used alone or in combination with trimethoprim in commercial eye drops  to treatImportantly, MIPs can enhance the load and duration of release of lipophilic and/or high-molecular-weight drugs compared to the low affinity of conventional, non-imprinted polymers. Although some MIP-based contact lenses are thicker than commercial contact lenses to achieve the therapeutic dose of the loaded drugs, the above cases of MIP-based contact lenses indicate their potential as potentially reusable vehicles for ocular drug delivery.Even though conventional polymer-based contact lenses may exhibit high bioavailability, their life span is limited ,97. By cBiologics and anti-tumor agents typically require regular injections/infusions to maintain the desired therapeutic effects ,99, and Paul et al. proposed a molecularly imprinted oral insulin DDS to evaluate the possibility of delivering therapeutic doses of insulin via the oral route  Figure . Insulin−1, whereas the BGL did not change after the oral administration of free insulin, insulin-loaded NIP NPs or blank MIP NPs.In another study, Paul et al. showed that the insulin loading capacity of the MIP NPs (~82%) was nearly three times higher than that of the corresponding NIP NPs . The in −1 and then maintained this level for up to 12 h. Although the relative pharmacological bioavailability of the insulin-loaded MIP NPs (~2%) was lower than that of subcutaneous injections of free insulin (100%), the enhanced loading capacity and sustained release of the MIP NPs made it possible to deliver therapeutic doses of insulin. This suggests the potential to avoid multiple daily injections of insulin—a regimen that often hinders compliance due to the associated inconvenience and discomfort.However, the insulin-loaded MIP NPs gradually reduced the initial BGL (~40% within 4 h) at a dose of 50 IU kgb-poly(lactide-co-glycolic acid) (PLGA) polymersome ..71].In both cases, the magnetic nanosystem is a “hot spot” that generates local hyperthermia when an external alternative magnetic field (AMF) is applied, thereby triggering the release of DOX. In in vitro drug release studies performed at 37 °C, the release of DOX from the MMIPs increased from 10% to 60% of the total payload after 4 h of exposure to the AMF via a mechanism of disrupted hydrogen bonds between DOX and the functional monomer. Similarly, the release of DOX from the magnetic nanogels increased from 24% to 45% after exposure to the AMF, but in this case, the increase in drug release was attributed to conformational changes in the gel network.Although the MMIPs released less total DOX (7 µM) than the nanogels (16.7 µM), they released more DOX within 4 h. In addition, after exposure to the AMF, both the MMIPs and nanogels decreased cancer cell viability . A key limitation of the MMIPs is their relatively low loading capacity compared to that of the nanogels, meaning that larger doses of MMIPs may be required in clinical practice. However, compared to the nanogels, MMIPs offer advantages in DOX release behavior, because they achieve specific binding between the drug and the imprinted cavities while nanogels rely on physical entrapment. Therefore, MMIPs may decrease DOX leakage in systemic circulation, thereby reducing the severity of the side effects.Another example of a DDS that responds to exogenous stimuli is the letrozole-loaded MMIP NPs (~100 nm in diameter) synthesized by Kazemi et al. for the treatment of breast cancer . The in Azidothymidine is an antiviral drug historically used in the treatment of AIDS and more recently shown to have direct anti-tumor activity . However−1) than the corresponding MNIPs (~38 mg g−1), MIP NPs (~46 mg g−1) and NIP NPs (~18 mg g−1). In an in vitro drug release study, the MMIPs released only 14% of the loaded drug at pH 7.4 but almost all of the drug at pH 5  cells. Although the effect of an AMF on the drug release behavior not assessed, the study concluded that MMIPs exhibit potential as pH-responsive vehicles to target drugs to tumors.N-isopropylacrylamide) [poly(NIPAM)] was exploited in another work for the controlled release of 5-FU. Li et al. proposed a temperature and magnetism bi-responsive MIP (TMMIP) that was obtained via surface-grafting copolymerization ..140].An epitope of the extracellular domain of EGFR and DOX were used as the template molecules. Flow cytometry was used to analyze the selective recognition capability for MIP NPs towards breast cancer cells overexpressing EGFR. Furthermore, DOX-loaded EGFR-MIP NPs decreased the cell viability of EGFR-overexpressing MDA-MB-468 cancer cells (lower than 75%) in comparison to free DOX (more than 90%) and control NPs (~100%). Moreover, the cell viability of non-EGFR-overexpressing cell-lines was virtually unaffected, highlighting the achievement of selective targeting and DOX delivery.Similarly, Qin et al. studied active targeting MIP NPs for the treatment of breast cancer. The MIP NPs were prepared with an epitope of the CD59 cell membrane glycoprotein and DOX as templates such that the high concentration of GSH and weak acid environment created by the cancer cells would selectively trigger DOX release  141]..141].−1 of the epitope, indicating the enhanced adsorption and strong specificity of the MIP NPs. After 15 days and in absence of GSH, about 50% of the drug leaked from pristine FZIF-8/DOX in PBS (pH 7.4), while almost no DOX was released from FZIF-8/DOX MIP NPs in PBS (at pH 5.0 and 7.4). In the presence of GSH, the DOX release from the MIP NPs greatly increased (at pH 5.0 and 6.0) to more than 90% over 15 days. In addition, the viability of MCF-7 cells (CD59 positive) was significantly lower after treatment with the FZIF-8/DOX MIP NPs  than after control treatments: FZIF-8/DOX NIP NPs (50%), FZIF-8/DOX (60%) and free DOX (60%). Importantly, normal cells did not show significant apoptosis; the anti-tumor effects were limited to the MCF-7 cancer cells. Moreover, the tumor volume (10 mm) in mice treated with FZIF-8/DOX MIP NPs was more than 2-fold smaller than that in mice treated with the controls. These results indicate that FZIF-8 MIP NPs can selectively target MCF-7 cells and trigger an adequate drug release in the presence of GSH, thereby reducing systemic toxicity and improving the therapeutic index of DOX.Fluorescent zeolitic imidazolate framework-8 (FZIF-8) was employed as the framework of the MIP NPs because FZIF-8 is fully biodegradable in an acidic environment. The fluorescence intensity of the MIP NPs was ~4-fold higher than that of the NIP NPs with 0.1 mg mLAs can be seen, in comparison to the conventional active targeting polymeric DDSs, double-imprinted MIP NPs offer a benefit by significantly reducing aspecific drug release and improving therapeutic effects. In addition, while biological ligands might exhibit superior specificity in certain instances, the high costs and complex production processes associated with materials such as antibodies and peptides may be prohibitive ,142,143.Double-imprinted polymeric DDSs, on the other hand, are extremely affordable. Solid-phase synthesis technology, in particular, makes it possible to recycle the templates depending on the production conditions, which greatly optimizes the resources whilst containing the costs . FurtherIn the last 5 years, a myriad of interesting studies has revealed great potential for the development of MIP-based DDSs. Many have focused on drug delivery for anti-tumor agents, which are important candidates for improved DDSs because the majority of these drugs possess a narrow therapeutic window. Methacrylic monomers and cross-linkers such as HEMA, MAA, MBA, EGDMA and EDMA have been widely used in the advancement of MIP-based DDSs ,145. The2 has a great potential for the development of highly pure, GMP-compliant MIP-based DDSs [Another major obstacle in the development of most MIP-based DDSs is the use of organic solvents  . These fsed DDSs .A further challenge that arises from the analysis of the above-discussed literature examples, is that only a limited number of studies reported in vivo data for the evaluation of MIP-based DDSs. In vivo research should be considered an indispensable step in the evaluation of any potential DDS. Since most studies evaluated toxicity and drug release only in vitro, further investigations and data are needed to ensure that the promising in vitro behavior of MIPs translates in in vivo models exhibiting safety and adequate release properties. Only through in vivo studies can we adequately evaluate the possible advantages of MIPs in clinical practice.Another relevant concern in some cases is the inadequate drug loading of MIP-based DDSs . This may result in sub-par, ineffective drug release. Importantly, though, even MIP-based DDSs with somewhat inadequate loading usually perform better than the corresponding NIP-based DDSs, indicating the potential advantages of molecular imprinting to enhance drug loading. The loading capacity may be increased by employing a smaller proportion of cross-linkers, but this strategy must also consider the trade-off between drug loading and the stability of imprinted cavities.Furthermore, some MIPs may undergo a small initial burst release due to the adsorption of drug molecules onto their surface. For some drugs , this may cause serious toxic effects. In other cases, however, burst release can be beneficial  . PerhapsConventional drug delivery can be burdened by systemic toxicity and low bioavailability because of non-specific delivery and rapid clearance. Recently, in attempts to address these limitations, a variety of MIPs have been evaluated as novel DDSs. An analysis of the literature on MIP-based DDSs reveals a great potential for extensive research, particularly for the delivery of drugs with narrow therapeutic windows and/or low bioavailability. Although the process of designing and translating MIP-based DDSs into clinical practice is still in its infancy, ongoing development will most likely lead to the creation of innovative drug delivery vehicles with commercial value.Imprinted polymers can not only significantly enhance the drug loading and the stability of drugs in harsh conditions but also attenuate the release behavior by engineering specific interactions between drug molecules and functional monomers. As an example of the advantages of rate-programmed drug delivery, MIPs as excipients can be used to develop sustained transdermal formulations, therapeutic contact lenses and oral formulations for protein delivery. In addition, MIP-based DDSs can be designed with stimuli-responsiveness, using properties of the imprinted cavities to achieve enhanced release profiles compared to conventional polymeric DDSs. Even more interestingly, active targeting drug delivery can be achieved via unique double-imprinting of targeting moieties and drugs.However, much of the extant research does not go so far as to include clinical studies that address biomedical regulations regarding the novel drug delivery devices. Although some studies report the in vitro cell toxicity of MIP-based DDSs, the therapeutic effects and clinical safety cannot be determined without in vivo assessments. Therefore, additional ongoing efforts are needed to design, develop and evaluate MIP-based DDSs to evaluate their safety profiles and satisfy biomedical regulations.With an eye on the horizon, a combination of MIP-based DDSs and implantable microchips has great potential for self-regulated drug delivery  as the n


The pandemic has affected all aspects of life and continues to spread despite the stringent control measures. Meanwhile, scientists all around the world have been scrambling to ascertain how the virus spreads and find out the effective ways to put this outbreak quickly under control, focusing on both persistent strict domestic interventions and vigilance against exogenous imported cases.6Furthermore, due to the widespread expansion of the coronavirus disease 2019 (COVID-19) epidemic around the world, different areas experienced a resurgence of COVID-19 cases after a relaxation of social distancing policies. However, there is an emerging concern regarding the reinfection in previously recovered SARS-CoV-2 patients. In former coronavirus pandemics, including the Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS), immunoglobulin levels in patients lasted for a minimum of 2 years, indicating that patients could be vulnerable to reinfection after 3 years. Emerging evidence suggested that SARS-CoV-2 could present similar behavior and reactivate in patients with previously confirmed COVID-19 infection and cause illness and person-to-person transmission.8.Herein, we report on 2 epidemiological conditions that evolved during the pandemic spread of novel coronavirus (COVID-19), within the Libyan communityThe first condition we report concerns the epidemiological features of a family cluster of 6 patients in Libya, as 1 of them had a history of travel to Istanbul and 5 other family members had not traveled anywhere. The demographic, epidemiological, and clinical features; chest radiography; laboratory tests; and outcomes were obtained for each patient. An asymptomatic case was defined as a laboratory-confirmed COVID-19 infection case who was afebrile and well.7 A total of 6 patients of the same family were transferred to a special COVID-19 center designated for treatment and isolation from September 13 to October 27 as illustrated in The second condition is regarding a case of reinfection from SARS-CoV-2 in the Libyan Community (8ommunity . A 52-yeommunity .7,8et al. and Mulder et al.10 However, further studies are needed to clarify whether this second infection is due to the acquisition of a new or more pathogenic strain, particularly after the emergence of a new virulent strain nowadays.During the second episode, on September 29, 2020, the patient was admitted again with fever, cough, shortness of breath, and gastrointestinal symptoms . The cli6Family clustering and reinfection represent a new clinical and epidemiological challenge in the COVID-19 pandemic. Therefore, policy planning should consider these contributing factors. Family clusters were common in the development of this epidemic. Hence, special caution is needed for asymptomatic family members, and policies should be developed to forbid large-scale family meetings and unnecessary visits. Furthermore, reinfection findings may have certain implications whether the reinfection occurred with the same stain or a newly emerging one. This highlights the role of vaccination in response to COVID-19. If we have truly reported a case of reinfection, initial exposure to SARS-CoV-2 might not result in a level of immunity that is defiantly protective for all individuals indicating similar perspectives to the influenza vaccine. However, initial genome analysis studies showed that the SARS-CoV-2 strains from the first and second episode in our patient belong to different clades/lineages with 24 nucleotide differences, suggesting that the virus strain detected in the second episode is completely different from the strain found in the first episode.


Ferroptosis is a newly discovered form of cell death characterized by iron-dependent lipid peroxidation. This study aims to investigate the potential correlation between ferroptosis and the prognosis of lung adenocarcinoma (LUAD).RNA-seq data were collected from the LUAD dataset of The Cancer Genome Atlas (TCGA) database. Based on ferroptosis-related genes, differentially expressed genes (DEGs) between LUAD and paracancerous specimens were identified. The univariate Cox regression analysis was performed to screen key genes associated with the prognosis of LUAD. LUAD patients were divided into the training set and validation set. Then, we screened out key genes and built a prognostic prediction model involving 5 genes using the least absolute shrinkage and selection operator (LASSO) regression with tenfold cross-validation and the multivariate Cox regression analysis. After dividing LUAD patients based on the median level of risk score as cut-off value, the generated prognostic prediction model was validated in the validation set. Moreover, we analyzed the somatic mutations, and estimated the scores of immune infiltration in the high-risk and low-risk groups. Functional enrichment analysis of DEGs was performed as well.High-risk scores indicated the worse prognosis of LUAD. The maximum area under curve (AUC) of the training set and the validation set in this study was 0.7 and 0.69, respectively. Moreover, we integrated the age, gender, and tumor stage to construct the composite nomogram. The charts indicated that the AUC of LUAD cases with the survival time of 1, 3 and 5 years was 0.698, 0.71 and 0.73, respectively. In addition, the mutation frequency of LUAD patients in the high-risk group was significantly higher than that in the low-risk group. Simultaneously, DEGs were mainly enriched in ferroptosis-related pathways by analyzing the functional results.This study constructs a novel LUAD prognosis prediction model involving 5 ferroptosis-related genes, which can be used as a promising tool for decision-making of clinical therapeutic strategies of LUAD.The online version contains supplementary material available at 10.1186/s12890-021-01588-2. The ferroptosis is an iron-dependent form of regulated cell death (RCD) that has been recently discovered. It differs from the apoptosis, necrosis, and autophagy . The impLung cancer is the most prevalent one among malignancies and it is the chief leading cause of tumor-related deaths worldwide. Pathologically categorized, about 85% of lung cancer cases belong to NSCLC, of which lung adenocarcinoma (LUAD) is one of the most frequent subtypes . BesidesRNA sequencing data of ferroptosis-related genes and clinical information of LUAD patients were downloaded from the public databases. It is shown that expression levels of ferroptosis-related genes were correlated to survival outcomes of LUAD. In the present study, LUAD patients were divided into a training set and a validation set based on the random stratified sampling of tumor stages. Then, we established the multi-gene LUAD prognosis prediction model and calculated risk scores through the LASSO regression with tenfold cross-validation, and univariate and multivariate Cox regression analyses. Finally, the established model was verified in the validation set and the overall sample set, aiming to test the fitting degree of the model. To explore the underlying molecular mechanism of the difference in the prognosis of LUAD, we further performed immune and biological functional enrichment analyses.https://portal.gdc.cancer.gov/) involved 533 cancer specimens and 59 paracancerous ones. Their raw RNA-Seq data, single-nucleotide variation (SNV) data and clinical information were included as well. The mRNA expression data were normalized using the DESeq2 variance stabilizing transformation (VST). Based on the previous research, we selected the top 60 ferroptosis-related genes as the candidate gene set  procedure. Genes with HR ≠ 1 and p < 0.05 were selected as prognosis-related genes. Subsequently, the intersection set between the identified DEGs and prognosis-related genes was taken, and their downstream functions in influencing the prognosis of LUAD were further analyzed.The DESeq2 package in R  was utilThe glmnet package in R was utilized to perform LASSO regression analysis on the target gene set  and the To explore the survival predictive feasibility of risk scores obtained from the multi-gene prognostic prediction model, we used the "survival" and "survminer" packages and the Kaplan–Meier method to estimate the survival curve. The time-dependent ROC curve was plotted by the "survival ROC" package in G, which graphically displayed the predictive ability in the prognosis of LUAD at different time points.To establish a more reliable prediction method that could be applied in clinical practice, we constructed the composite nomogram through the rms package. Similarly, clinical features and risk scores of LUAD patients were combined to establish a multivariate Cox regression model. Based on the influenced degrees of risk factors on the outcome, they were graded to value its level, and a total score was obtained by the sum of the score of each factor. Finally, through the converse relation between the total scores and the outcome events, the predicted survival time of LUAD patients was calculated.We further verified the accuracy of the nomogram by applying the model using the C-statistic, calibration curve and time-dependent ROC curve. Meanwhile, the nomogram model was calibrated to predict the proximity between the predicted survival and the actual result numerically. The Hosmer–Lemeshow (H–L) test was performed by dividing data into 3 ascending ordered groups (tertiles) based on the predicted result obtained from the model, thus testing the goodness of fit for the χ2 test. Furthermore, the average predicted survival was compared with the actual event rate estimated using the Kaplan–Meier method.DEGs between high-risk groups and low-risk groups divided by risk scores were obtained. Subsequently, enrichment analysis of gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of DEGs were analyzed using the clusterprofiler package in R .The single-sample gene set enrichment analysis (ssGSEA) on 29 immune gene sets  involvinThe sample function in R was used to perform the random stratified sampling. Meanwhile, the heatmap of DEGs and SNV mutation landscape were respectively depicted by the pheatmap and maftools packages in R . Wilcoxoadj p < 0.05). Taking the intersection of the 11 genes and DEGs, it is obviously shown that the former ones were differentially expressed between LUAD and paracancerous specimens. In addition, the results of Cox regression analysis were depicted in the forest plots , GSS, ACSL3 and PGD genes were the risk factors for the prognosis of LUAD, while the PEBP1 gene was a protective factor (adj P < 0.05) . Later, the predictive potential of the risk score model in LUAD was assessed by depicting the ROC curve, and the highest AUC was 0.7  and validation set (n = 251). Meanwhile, LASSO-Cox regression analysis of the above-mentioned set was performed to identify prognosis-related genes that were correlated to ferroptosis Fig. a, b. The and valip = 0.00042), and the maximum AUC was 0.69 , and the maximum AUC was 0.69.To determine the reliability of the established model and the predictive accuracy, results obtained from the prognostic model were validated in the verification set. Similarly, risk scores in the validation set, and subgroup classification of LUAD patients into the high-risk group and low-risk group with the same threshold (cut-off = 6.64) were conducted. Moreover, the survival status of LUAD patients in the validation set was estimated using the KM algorithm, followed by plotting the survival curve. It is consistently shown that the prognosis of LUAD patients in the high-risk group was significantly worse than that of the low-risk group  and risk score as variables in the multivariate Cox regression analysis, and incorporating the above factors into the model as variables, a nomogram was obtained Fig. a, in whiTP53 (53%), TTN (50%), MUC16 (42%), CSMD3 (40%), and RYR2 (39%)].We downloaded the SNV data of 502 LUAD patients, and subgrouped based on the risk score. Each figure of the mutation landscape indicated the top 20 genes with the highest mutation frequency, and their mutations types were labeled with different colors Fig. . The uppTo further explore the differences in biological functions and pathways between the high-risk group and low-risk group, the DESeq2 package was used to analyze expression levels of DEGs between the high-risk group and low-risk group . DEGs screened out from both groups were later subjected to GO and KEGG enrichment analysis. The results of pathway enrichment and GO enrichment were shown in Fig. p < 0.05). Besides, the enrichment scores of these immune function gene sets in HLA, inflammation promoting, MHC class I, T cell co-stimulation and type II IFN response were also significantly different between groups (p < 0.05).According to the threshold of risk score, the correlation between risk score and immune status in LUAD patients was further explored. We obtained a total of 29 immune-related gene sets involving multiple immune cells and immune-related functions or pathways. To quantify the enrichment degree of transcriptome data in the 29 immune gene sets, separate enrichment scores for each paring of a sample between the high-risk risk and low-risk group were estimated by the ssGSEA and compared by depicting a boxplot  of LUAD. In this study, the LUAD dataset was divided into the training set and validation set by the random stratified sampling method. The prognostic prediction model involving 5 genes was established for the training set samples through the LASSO regression with tenfold cross-validation and univariate and multivariate Cox regression analysis, which was validated in the actual clinical practice.Previous evidences have confirmed the vital functions of ferroptosis-related genes in the entire ferroptosis process. Nevertheless, the specific influence of a single ferroptosis-related gene on the prognosis of a certain type of cancer remains unclear. We combined the mRNA expression levels of each gene with actual clinical characteristics to analyze the differential expression between LUAD and paracancerous specimens. Moreover, quantitative data of gene expressions were analyzed for their predictive potential in the survival of LUAD. Subgrouped by the risk score, identified genes between the high-risk group and low-risk group were analyzed for their differential variations, biological functions and pathways they were mainly enriched in. In conclusion, our screened DEGs in LUAD patients may be potential targets involved in the occurrence and development of LUAD.ACSL3, ACSL4, GSS, PEBP1, PGD) were involved in, which were closely associated with the process of ferroptosis. The ACSL3 gene is responsible for exogenous monounsaturated fatty acids to protect cells against ferroptosis, and it is negatively correlated with ferroptosis sensitivity [ACSL4 gene is essential for proferroptosis. Knockdown of ACSL4 inhibits erastin-induced ferroptosis, and its overexpression can restore ferroptosis sensitization. Re-expression of flag-tagged human wild-type (WT) ACSL4 (ACSL4-Flag) in Acsl4 KO (Acsl4−/−) Pfa1 cells restores full sensitivity to ferroptosis induction, and knockdown of it significantly prolongs survival compared to vehicle-treated mice. Knockout of ACSL4 in ferroptosis-sensitive cells protects erastin- and RSL3-induced cell death [GSS gene provides instructions for making glutathione synthetase. The glutathione-dependent lipid hydroperoxidase GPX4 contributes to prevent ferroptosis by converting lipid hydroperoxides into non-toxic lipid alcohols. Overexpression of PEBP1 increases the sensitivity of HK2 cells to RSL3, and knockdown of PEBP1 in HAEC and HT22 cells yields an opposite result [PGD gene is involved in erastin-induced ferroptosis [In the constructed predictive model, a total of 5 genes , TTN (50%), MUC16 (42%), CSMD3 (40%) and RYR2 (39%) genes presented 5–13% higher mutation frequency than those in the low-risk group. Calculating the mutation frequency easily identified the source of the variations between groups. Genomic analyses about the prognosis difference between high-risk and low-risk LUAD patients need to be performed in the future.To explore the factors for the prognosis difference between the high-risk group and low-risk group, we compared the SNV levels and analyzed the differences in biological functions of the two groups. After subgrouping the acquired SNV statistics according to high-risk and low-risk scores, the mutation frequency of each gene and the number of mutations in each sample were calculated, which were displayed as the mutation landscape. The results revealed that LUAD patients in the high-risk group presented a higher frequency of each gene mutation. Moreover, the top 5 genes with the highest mutation frequency in the high-risk group, involving the staphylococcus aureus by establishing efficient iron transportation systems. On the one hand, staphylococcus aureus consumes hemoglobin in the red blood cells of the host and serum through the transport systems encoded by the iron-regulated surface determinants located in cell wall. On the other hand, it acquires the iron by the siderophores with a high affinity for iron. A previous study has shown that the reactive oxygen species can result in the drug resistance in staphylococcus aureus infection [Functional enrichment analysis and immune infiltration score on DEGs between the two groups were further performed. DEGs were mainly enriched in the cytochrome P450, steroid hormones, IL-17 signaling pathways, staphylococcus aureus infection pathways, etc. The cytochrome P450 oxidoreductase (POR) is necessary for triggering the ferroptosis in cancer cells . Moreovenfection . Previounfection , 31. TheAlthough constructing a prognostic model is of great significance in the TCGA-LUAD cohort, only internal verification and overall verification were performed in the verifying process of the model. Specifically, the external validation set was not included in the examination of the final results. In functional analysis, enrichment analysis was performed in DEGs between the high-risk group and low-risk group, while functions of these pathways were not be experimentally verified. As a result, we only identified which pathways and functions were responsible for triggering the prognosis difference in high-risk and low-risk LUAD patients, but how they induced it needs to be further explored.In general, we established a prognostic prediction model based on 5 ferroptosis-related genes by analyzing the LUAD dataset. In the training and validation set, this model was found effectively predict the overall survival of LUAD, and more important, clinical features of LUAD patients were taken into consideration, which remarkably simulated the actual clinical practice. Our findings provided a promising tool in predicting the prognosis of LUAD patients, and theoretical references for explaining the prognosis difference between high-risk and low-risk LUAD.This study constructed a novel LUAD prognosis prediction model based on 5 ferroptosis-related genes, which can provide a reliable prognostic evaluation tool for clinical practice and assist the clinical therapeutic decision.Additional file 1:Supplementary figure 1. COX regression PH hypothesis test for each candidate.Additional file 2: Supplementary figure 2. Gene expression threshold setting. Additional file 3. Supplementary figure legendsAdditional file 4. Supplementary table legendsAdditional file 5: Supplementary table 1. Ferroptosis-related genes setAdditional file 6: Supplementary table 2. Gene list of immune cells and immune-related functions.Additional file 7: Supplementary table 3. GO enrichment results of DEGs between high risk group and low risk group.Additional file 8: Supplementary table 4. Pathway enrichment results of DEGs between high risk group and low risk group.


In all three cases, the STEM/PNBD results were comparable to those obtained from TEM/SAED. Therefore, the 4D-STEM/PNBD method enables fast and simple analysis of nanocrystalline materials, which opens quite new possibilities in the field of SEM.We introduce a novel scanning electron microscopy (SEM) method which yields powder electron diffraction patterns. The only requirement is that the SEM microscope must be equipped with a pixelated detector of transmitted electrons. The pixelated detectors for SEM have been commercialized recently. They can be used routinely to collect a high number of electron diffraction patterns from individual nanocrystals and/or locations , as we obtain two-dimensional (2D) information for each pixel of the 2D scanning array). Nevertheless, the individual 4D-STEM diffractograms are difficult to analyze due to the random orientation of nanocrystalline material. In our method, all individual diffractograms  are combined into one composite diffraction pattern . The final powder diffraction pattern can be analyzed by means of standard programs for TEM/SAED (Selected-Area Electron Diffraction). We called our new method 4D-STEM/PNBD (Powder NanoBeam Diffraction) and applied it to three different systems: Au nano-islands , small TbF Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) are well-established imaging and analytical techniques for characterization of surfaces and thin samples, with numerous applications in many fields of material and life sciences. Typical SEM microscopes use electrons with an energy of 30 keV and below, while typical TEM microscopes operate in a higher energy range between 60 and 300 keV. In both SEM and TEM, it is possible to detect transmitted electrons by means of a technique called scanning transmission electron microscopy (STEM) . The SEMFurther development in the field of STEM imaging headed towards multi-segmental annular detectors and, subsequently, to 2D pixel array detectors . Multi-segmental STEM detectors consist of a central segment in the BF region and several annular (or semi-annular) segments in ADF and HAADF regions . InThe pixelated STEM detectors record intensity of the scattered electrons at given primary beam position as a function of their direction or, in other words, they record a 2D diffraction pattern at current beam position. If the scanning beam is narrow (and not convergent), the probe size is small and the recorded patterns correspond to nanobeam diffraction (NBD) known from TEM . As theFrom the point of view of everyday routine use, all above-listed applications of 4D-STEM microscopy have two limitations: At first, the great majority of 4D-STEM methods were developed and tested on TEM. Nevertheless, even simplified versions of these methods would substantially enhance the analytical possibilities of SEM microscopes, where the classical diffraction methods, such as selected-area electron diffraction (TEM/SAED), are unavailable. Second, all above-listed 4D-STEM methods focus on analysis of individual diffraction patterns, which is usually complex and requires detailed crystallographic knowledge together with specialized software. The complexity consists in that the analyzed three-dimensional (3D) nanocrystalline materials are  oriented randomly and, as a result, we record randomly oriented sections of their 3D diffractograms.In this work, we introduce a novel, fast, and simple 4D-STEM diffraction technique, which can be employed in any SEM microscope equipped with a pixelated STEM detector. The method does not try to analyze individual diffraction patterns from every single nanocrystal and/or location but combines the whole 4D-STEM dataset into one composite diffraction pattern that is completely analogous to TEM/SAED diffractogram of a polycrystalline sample. We called the new method 4D-STEM/PNBD (Powder NanoBeam Diffraction), as the resulting diffractogram is a combination of the individual NBD patterns. The great advantage of our simplified approach consists in that the 4D-STEM/PNBD polycrystalline diffraction patterns are much easier to process than the whole dataset of individual diffractograms. This makes the method accessible to all experienced users, not only to professional crystallographers. Moreover, the processing of 4D-STEM/PNBD polycrystalline diffraction patterns does not require specialized, heavy-weight crystallographic packages. The summation of the individual diffractograms can be performed in a few minutes by means of quite simple and easy-to-adjust scripts in freeware Python programming language (these scripts are currently available upon request from the first author). The processing of the final 4D-STEM/PNBD diffractogram can be performed by any small, simple, and stand-alone program for analysis of TEM/SAED powder diffraction patterns (such as ProcessDiffraction ). The ne3 nanocrystals (size < 5 nm), and the third sample consisted of NaYF4 nanocrystals (size > 100 nm). The preparation of the samples was described in detail in our previous studies  = [001] and the strongest experimentally observed diffraction peaks were of hk0 type .3 dataset, which is used as an example in this section, contained 100 × 80 = 8000 positions . All modules are part of many Python distributions . Therefore, usage of STEMDIFF is extremely easy: after installing a suitable freeware Python distribution that contains the necessary modules (such as WinPython or Anaconda), it is enough to download the scripts (at the moment they are available upon request from the first author of this work) and apply them to a given dataset. Another advantage of our approach relying on standard scientific modules consists in that the underlying algorithms of the modules are highly optimized, typically written in C or Fortran and, as a result, the STEMDIFF scripts are very fast. Even the processing of the largest datasets (>2.5 GB of data) took less than 3 min with a common modern computer.sum-all.py) and save the resulting file after suitable rescaling as a grayscale diffraction pattern image  and agglomerated . For such cases, the STEMDIFF package enables the automatic filtering of the dataset and summation of the files containing strong diffraction spots, while the files containing noisy information  are excluded. At first, the dataset is visualized by means of script inspect-data.py , which then sums only the data files with strong diffraction spots . After rescaling , the final diffractogram is saved as a grayscale image  on the quality of final 4D-STEM/PNBD diffraction patterns was small. The radially averaged intensity of 4D-STEM/PNBD diffractograms showed negligible changes with increasing size of the dataset . All datAu big , that coAu big a,b and rAu big c were caAu big . The dif scripts b, while y script c. The 4Dy script a and shoy script b displayy script c. This eAu small , and whysum-all.py, sum-selected.py, rescale.py, and rad-ave.py), as shown schematically in The effect of the dataset filtering  was strong and important. The results for all three samples are summarized in S > 7.7) had 22,241 files, and the filtered dataset containing just the files with strong diffraction spots (S > 8.0) comprised 3485 files. The results for dataset Au small , their size was close to ideal , and they cover the carbon film in a homogeneous layer (which minimized the amount of data files containing only background noise). Consequently, the Au dataset exhibited intensive diffractions even without any modification . Moreover, the TbF3 nanocrystals tended to form agglomerates, which led to the empty areas on the supporting carbon film, and the diffractograms of the carbon film just increased the background noise. As a result, the unfiltered dataset  was used for the final comparison with TEM/SAED and PXRD results and good agreement was achieved as well, as documented in The small TbF4 nanocrystals were the most difficult sample for 4D-STEM/PNBD: the crystals were transparent for 120 keV electrons in TEM/SAED . The 4D-STEM/PNBD diffractogram calculated from the dataset without filtering showed virtually no peaks, because a great majority of the diffractograms contained a lot of background noise  was not easy due to low-intensity, broad diffraction peaks  in the whole range of radial distances and diffraction intensities , while our contribution is based on a DED-based pixelated STEM detector .The main difference between the 4D-STEM/PNBD method and all other 4D-STEM techniques described in the literature consists in that that 4D-STEM/PNBD intentionally avoids processing of the individual diffractograms  and converts the 4D-STEM data cube into one simple powder electron diffraction pattern (that can be processed easily with exactly the same simple programs that are used for TEM/SAED powder diffractograms). Nevertheless, the possibility to convert the whole 4D-STEM dataset into one powder electron diffractogram did not escape the attention of other researchers completely, although their studies were focused on different 4D-STEM data processing strategies. The detailed review dealing with basic principles and applications of 4D-STEM methods in TEM  summarizThe most important limitation of the current version of the 4D-STEM/PNBD method consists in high background that may be difficult to remove in the case of low-intensity diffraction peaks. We have demonstrated that the influence of background can be suppressed significantly by dataset filtering . Neverth3, The second limitation is an intrinsic property of the 4D-STEM/PNBD method: by combining all diffractograms into one composite diffraction pattern, we lose the local information about the orientation of individual crystallites. Nevertheless, the method is still able to probe quite small areas on the investigated specimens. Considering that it is perfectly enough to collect around 8000 data files even for samples containing quite small crystallites  yielded good results. Even the largest nanocrystals  exhibited diffraction intensity sufficient for further processing, although the diffraction peaks were close to the detection limit, as illustrated in The third limitation is the maximum thickness of the samples that can be studied by the 4D-STEM/PNBD method. The situation is analogous to TEM/SAED: the crystals should be thin enough so that a sufficient number of diffracted electrons penetrate through the specimen. The penetration depth of electrons into a specimen decreases with the accelerating voltage. The maximum accelerating voltage in a great majority of SEM microscopes is 30 kV , while the accelerating voltages in typical TEM microscopes range from 60 to 300 kV. Consequently, the thickness of the nanocrystals for 4D-STEM/PNBD should be even lower than in the case of TEM/SAED, but our results suggested that this limitation is not critical. The small- and medium-sized nanoparticles . In fact, the microscope used in this study  was a dual-beam microscope containing both electron and ion beam columns. The combination of FIB and pixelated STEM detector in one device enables to prepare thin lamellae from bulk specimens and characterize them routinely, by means of the fast and straightforward 4D-STEM/PNBD method.