Lesbian, gay, bisexual, and transgendered (LGBT) people are marginalized and understudied. Analyzing research activity worldwide is vital to better understand their needs in confronting the HIV epidemic. This study aimed to evaluate the global literature to identify the research collaboration, content, and tendency in HIV-related issues among the LGBT populations.Peer-reviewed original articles and reviews were achieved from the Web of Science Core Collection database. Country’s collaborations and co-occurrence of most frequent terms were illustrated by VOSviewer software. The Latent Dirichlet Allocation (LDA) and the linear regression model were utilized to uncover the hidden topics and examine the research trend.From 1990 to 2019, a total of 13,096 publications were found. Stigma, sexual risk behaviors and HIV testing were the major topics in the LGBT research during the study period. Among 15 topics, topics about HIV/Sexually transmitted infections (STIs) prevalence, Outcomes of HIV/AIDS care and treatment, and Opportunistic infections in HIV-positive LGBT people showed decreasing attention over years, while other topics had a slight to moderate increase.Our study underlined the exponential growth of publications on the LGBT population in HIV research, and suggested the importance of performing regional collaborations in improving research capacity. Moreover, further research should focus on examining the manner to increase the coverage of HIV testing and treatment, as well as implement HIV-interventions with low cost and easy to scale-up. SinAlthough homosexuality is no longer classified as an illness , it doesSubstantial activities to advocate the rights of LGBT populations and alleviate the social stigmatization toward non-heterosexual groups have been developed in the last decades . One imp2.2.1.The Web of Science Core Collection was used to search the publications. This database was selected based on its advantages in providing more comprehensive data compared to other databases such as Scopus or PubMed . We set In this study, we conducted a search process in two phases. First, we searched the articles about HIV/AIDS by using the topic terms “HIV,” “AIDS,” “Human Immunodeficiency Virus,” and “Acquired Immune Deficiency Syndrome.” Then, with the HIV/AIDS-related dataset, we used the following terms for searching in title/abstract in order to filter studies about the LGBT populations:Terms for lesbian: lesbian, women who have sex with women, WSW, homosexual female, lesbianismTerms for bisexuality: bisexual, bisexualityTerms for transgender: transgender, transgendered, transsexualism, transsexual, gender identity disorders, gender dysphoriaTerms for gay: men who have sex with men, MSM, gayGeneral terms: LGBT, homosexuality, homosexual, same-sex, queer, sexual minority, not exclusively heterosexual.2.2.Information of selected documents was analyzed and presented including the year of publications, the number of publications per year, total citation up to 2019, mean citation rate per year, total usage in the last 6 months/5 years, and mean use rate in the last 6 months/5 years. The VOSviewer  software was employed to visualize the co-occurrence of most frequent terms and the country’s collaboration networks. Stata 15.0 was used for topic modelling and research trend identification.Latent Dirichlet Allocation (LDA) was employed to examine the contents of 15 latent topics. The LDA is processed based on the text mining principle, which helps to identify the topics among the unstructured body of texts. It is an algorithm that allows grouping a set of words with similar meanings to reflect a specific topic. For example, we assumed that articles on the topic of PWIDs would have the words “injection” or “drug users,” while the words “female,” “sexual,” and “workers” would be appeared more frequently in the articles on the topic of CSW. Topic modelling enables us to define the particular number of topics first, and then extract and allocate the words under these topics. After obtaining the outputs of the LDA model, we labelled the topics by referring two sources: (1) the top 15 words (after sorted) with the highest probability within each topic; and (2) titles/abstracts of publications within each topic. HIV and RDS experts are invited to label the topics in order to provide the most semantically meaningful interpretation.p was less than 0.05.We also calculate the volume and share of publications per topic per year. A linear regression model was conducted to detect the trend of each research topic in different time periods , with the share of publication as an outcome variable, and the number of years as a predictor variable. “Hot’ topic was defined when the coefficient was significantly positive, and the “cold” topic was detected when the coefficient was significantly negative. Statistical significance was identified if the value of 3.Analysis of the most frequent terms is visualized in via publication share (%) is visualized in Results of topic modelling by using the LDA technique are revealed in In the 90s decade, the topics about HIV testing (topic 2), molecular epidemiology of different types of HIV (topic 3), substance use (topic 5), inequalities in HIV-related risk behaviors (topic 7), socio-legal aspects of homosexuality (topic 8), pre-exposure prophylaxis (topic 12), and stigma-related barriers (topic 14) showed the positive trend, while the topics about HIV/STI prevalence (topic 1), and opportunistic infections (topic 10) had the negative tendency. From 2001 to 2010, topics 2 and 7 remained the positive trend, along with topic 9  and topic 13 ; whereas the publication share of topic 1, topic 6 (HIV treatment outcomes), and topic 10 continuously decreased over this period. During 2011–2019, topics 1, 3, and 4  became “cold” topics, while topics 11 , 12, and 14 were more popular. Overall, topics 1, 6, 10 showed a negative trend, while topics 2, 3, 5, 7, 8, 9, 11, 12, 14, and 15 showed a positive trend .4.This study evaluated the development of publications on the LGBT populations in the field of HIV/AIDS in the period from 1990 to 2019. Study findings indicated remarkable research productivity in this area over time, which focused mainly on gay or men who have sex with men groups, with the major topics about HIV-related risk behaviors, prevention and treatment service access, stigma, and mental health. Moreover, by analyzing the research topics, we found the change of research focus in the different time intervals, which can be useful for proposing future research agenda.Our analysis indicated the rapid expansion of publications studying HIV-related issues in the LGBT groups from the early 90s, one decade after the first AIDS case was detected . This suBy analyzing the growth of publications in different groups, we found that research on gay or MSM outweighed other populations, or in other words, global scholars paid more attention to MSM compared with lesbian, bisexual, or transgender people. This finding is understandable since the first AIDS record was related to MSM, and they are also one of the most-at-risk population for HIV infection due to unprotected anal sex practice, multiple sex partners, high frequency of substance use, large sexual network size, and highly stigmatized . In termvia articles with the most citations; whereas our advantage is the application of LDA, an unsupervised machine learning technique, to take into account all 13,096 papers to explore latent topics. Our results were also different from a bibliometric analysis in general LGBT studies, which found that social sciences were the main research topics, following by medicine and law  and NUS iHeathtech Other Operating Expenses (R-722-000-004-731).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Analyzing the whole transcriptome by RNA-seq data, we found that HsfA1a mainly regulated the genes involved in oxidative stress protection, protein homeostasis regulation and protein modification, as well as the response to biological stress in anthers under heat stress. The accumulation of reactive oxygen species in anthers was enhanced in hsfA1a mutants but decreased in HsfA1a-overexpressing lines. Furthermore, HsfA1a bound to the promoter region of genes involved in redox regulation , protein repair  and degradation  and regulated the expression of these genes in tomato anthers under heat stress. Our findings suggest that HsfA1a maintains pollen thermotolerance and cellular homeostasis by enhancing antioxidant capacity and protein repair and degradation, ultimately improving pollen viability and fertility.The heat shock transcription factors (Hsfs) play critical roles in plant responses to abiotic stresses. However, the mechanism of Hsfs in the regulation of pollen thermotolerance and their specific biological functions and signaling remain unclear. Herein, we demonstrate that HsfA1a played a key role in tomato pollen thermotolerance. Pollen thermotolerance was reduced in  HsfA1a promotes tomato pollen vigor and germination rate under heat stress by transcriptionally regulating the genes involved in antioxidant capacity, protein repair, and degradation capacity in tomato anthers.Global warming has led to frequent extreme weather, causing huge agricultural production losses every year. The male reproductive organs of plants are particularly sensitive to temperature changes. Heat stress could cause a series of effects on plant pollen in terms of cell structure, physiological metabolism, and molecular mechanism, and ultimately inhibit pollen germination and pollen tube elongation, reduce fruit setting rate, and lead to a decline in yield and quality , 2. TherArabidopsis [Arabidopsis, knockout of the HsfA5 homolog gene AtREN1 results in a defective HSR, leading to a high percentage of abnormal pollen grains [HsfA2, HsfA3, HsfB1, and HsfB2b of the Hsf family are upregulated after heat stress exposure in developing and mature pollen, whereas HsfA4c and HsfA5 remain unaffected [HsfB1 occurs in post-meiotic pollen and mature pollen, while HsfB2b is present in pollen in tetrads and during post-meiosis. HsfA3 and HsfA7 are much more stage-specific and are expressed in tetrads and the post-meiotic stage, respectively [HSFC1b is also responsive to high temperature, and heterologous expression of the Festuca arundinacea FaHsfC1b gene can improve Arabidopsis survival under heat stress [To counteract the damage caused by heat stress and maintain cellular homeostasis, pollen activates a heat stress response (HSR) depending on the intensity and duration of high temperature . The basbidopsis , 4. Accobidopsis . Class An grains . The HSRaffected . In tomat stress . These sPuGSTU17 in Populus ussuriensis to reduce ROS acumination and promote excess Zn tolerance [CaHsfA1d in Arabidopsis enhanced the expression of Hsfs and the antioxidant gene AtGSTU5, thereby maintaining H2O2 homeostasis and improving plant thermotolerance [Oxidative stress induced by environmental stimuli leads to the overproduction of reactive oxygen species (ROS), which often affects metabolic homeostasis, disrupting protein stability and membrane fluidity and ultimately causing pollen abortion . Plants olerance . Heterololerance . HoweverCellular PQC is one of the most essential biological processes under stresses. Stress-induced misfolded, damaged, and truncated proteins are rescued or degraded by PQC , 16. HSPrpt2a encoding the 26S proteasomal subunit in Arabidopsis showed impaired pollen development and delayed flowering [Arabidopsis are involved in pollen tube development, and lack of them results in defects in pollen germination [autophagy-related gene 6 (ATG6) in Arabidopsis resulted in defective pollen germination [ATG2-, ATG5-, and ATG7-RNAi lines of Nicotiana tabacum exhibit reduced pollen germination [atg7 mutants exhibit limited anther dehiscence, abnormal autophagosome formation, and reduced rice pollen numbers and fertility [Arabidopsis, heat stress promotes autophagy and autolysosome formation in wild-type (WT) anther wall cells and microspores, whereas abnormal tapetum degradation, reduced pollen grains, and defective anther dehiscence have been identified in atg5 mutants after heat stress [In addition to protein repair, protein degradation mechanisms are actively involved in plant responses to abiotic stresses. The ubiquitin–proteasome system (UPS) and autophagy are two critical pathways for protein degradation –20. The lowering . The ubimination . Furthermination . Rice atertility . Recent ertility , 17. Theertility . During t stress .Arabidopsis are functionally redundant [HsfA1a-overexpressing and knockout plants to study the HsfA1a-mediated thermotolerance mechanism in pollen. Transcriptome analysis results show that HsfA1a-induced gene expression is involved in the antioxidant system, the protein repair system, and the protein degradation and regeneration system. Moreover, HsfA1a can scavenge ROS accumulation and transcriptionally regulate the abundance of genes that are involved in redox regulation, HSPs, the UPS and autophagy in tomato anthers under heat stress. Therefore, this study provides important information for the regulatory networks and potential applications of HsfA1a in tomato pollen thermotolerance.The HsfA1 subfamily is defined as the main regulatory factor in response to high-temperature stress. The four HsfA1s AtHsfA1a, b, d, and e in edundant , 30. In edundant . In thishsfA1a mutants and HsfA1a-overexpressing (HsfA1a-OE) plants under heat stress. As shown in hsfA1a mutants under normal conditions, but was similar in HsfA1a-OE plants compared with WT as revealed by fluorescein diacetate (FDA) staining and aniline blue staining. Importantly, pollen vigor was reduced by 32.3 and 36.7% in hsfA1a-2 and hsfA1a-3 mutants, while it was increased by 48.3 and 59.2% in two lines of HsfA1a-OE plants under heat stress, respectively  or treated at 39°C (heat stress) for 3 hours. The sequencing data statistics are listed in Supplementary Data hsfA1a mutant anthers showed that the samples were segregated along PC1 with and without heat stress  are shown in To analyze the global transcriptional alterations caused by HsfA1a in anthers, we selected WT and  mutants . Howevert stress . Results mutants . These r2(FPKM) | >1 and P < 0.05 (where FPKM is fragments per kilobase of transcript per million mapped reads), including 25 antioxidant enzyme-related genes, 28 heat shock-related genes, 22 ubiquitin-related genes, 11 proteasome-related genes, and 8 ATGs, to further study the function of HsfA1a in transcriptional regulation of heat-response genes in tomato anthers  and nitroblue tetrazolium (NBT) staining to assay the accumulation of ROS and superoxide (O2•−) in anthers of different genotypes at the polarized microspore stage. The H2DCF-DA-stained ROS and NBT-stained O2•− in anthers kept signals at low levels under normal growth conditions in all genotypes.  could be bound by HsfA1a protein in vitro. When the HSE motifs of these five gene promoters were mutated, the HsfA1a binding probe signals were completely lost  Monoclonal antibody. 3HA-labeled HsfA1a transgene products were immunoprecipitated the in anthers of HsfA1a-OE5 plants after heat stress, but they were not enriched in WT plants. By contrast, the promoters of these three genes could not be pulled down by the IgG control antibody in anthers of both WT and HsfA1a-OE5 plants  maintained higher activity in the anthers of HsfA1a-OE plants under high temperature compared with WT  in vitro, whereas when the HSE motifs of the promoters of these six genes were mutated, binding to HsfA1a was completely lost  in heat-stressed anthers, and the results were similar to the expression trends of RNA-seq data  in vitro. The binding signals to HsfA1a were completely lost when the HSE motifs of these seven genes were mutated  under heat stress were consistent with the RNA-seq data  staining to detect autophagy activity in the tomato pollens. As shown in reatment . However with WT .hsfA1a mutants, WT, and HsfA1a-OE plants under normal temperature, but Atg8-PE bands were markedly enriched after heat stress  conjugates using western blotting and detected it with anti-Atg8a antibody as a marker for autophagy activation . The Atgt stress . InteresE plants . In our o plants . Then, wSE motif . The subT plants . We furtseq data 2D. UnderHeat stress can induce pollen HSR, leading to transcriptome, proteome, and metabolome reprogramming, which restores metabolism in pollen to maintain cellular homeostasis . Hsfs arArabidopsis myb21 mutant resulted in defective stamen development [MYB Important for Drought Response1 (MID1) in rice anthers increases POD and SOD transcript levels, thereby enhancing ROS scavenging ability to enhance drought tolerance [HsfA1a overexpression. As a major positive regulator of HSR, HsfA1a could transcriptionally regulate the expression of the GST8, MDAR1, and Cu/Zn SOD genes, thereby enhancing the activity of antioxidant enzymes and detoxifying ROS in tomato anthers under heat stress. These studies suggested that HsfA1a is closely related to the balanced regulation of ROS production and scavenging in pollen under heat stress.The antioxidant enzyme system is essential for regulating ROS production and the development of pollen and anthers . Howeverelopment . Overexpolerance . Our stuhos1 mutant plants [Arabidopsis AtUBP3/4 is involved in pollen tube development. The pollen of the ubp3 ubp4 mutant is incapable of undergoing mitosis II and exhibits pollen germination defects [UBP5 and UBP18) at high temperature was transcriptionally regulated by HSFA1a, which ultimately affected the accumulation of ubiquitinated proteins in anthers  plants [ATG10, ATG18d, ATG18g, and ATG18h, were induced at high temperature. Moreover, the number of punctate fluorescent signals of autophagosomes in tomato plant pollen was significantly increased after heat stress. In addition, studies have shown that ATGs can be induced by transcriptional regulation under different stresses. Brassinosteroid signaling can regulate ATG expression and autophagosome formation, which is important for coping with nitrogen starvation in tomato [ethylene response factor 5 (ERF5) transcriptionally regulates the expression of ATG8d and ATG18h and affects autophagy [ATG10 and ATG18f under drought stress [ATG10 could be transcriptionally regulated by HsfA1a. Meanwhile, we detected the accumulation of Atg8-PE protein in tomato anthers after heat stress, suggesting a potential role of autophagy in the regulation of heat tolerance in pollen and anthers.The UPS has a size limit on degraded molecules, and some oligomeric and aggregated proteins caused by stresses such as high temperature are too large to pass through the narrow proteasome entry channel . In cont) plants , 41. Hern tomato . Under dutophagy . In our In conclusion, we provide a comprehensive genetic and molecular analysis of tomato HsfA1a enhancing pollen heat stress resistance by upregulating antioxidant capacity, protein repair and degradation . We demoSolanum lycopersicum L. cv ‘Ailsa Craig’ was used in this study. We sowed seeds in trays with vermiculite and peat mixture (1:2/v:v). Three-week-old seedlings were transferred to plastic pots containing the same substrate. The conditions in the growth room were as follows: photoperiod was 12 hours/12 hours (light/dark), temperature was 25/20°C (day/night), and light intensity was 600 μmol m−2 s−1. For heat treatment, 8-week-old plants at the flowering stage were placed in a growth chamber at 39°C for 3 hours, and control plants were placed at 25°C for 3 hours. Six replicates were used in each treatment.hsfA1a mutants, a CRISPR-associated 9 (Cas9) vector was constructed according to the previously reported method [HsfA1a (GGAAGCCATCATGGAGGGAA) was designed using the CRISPR rgenome tools (http://www.rgenome.net/). Single-guide RNA (sgRNA) was annealed into a double strand and inserted into the AtU6-sgRNA-AtUBQ-Cas9 vector. The recombinant vector was confirmed by sequencing. After digestion with KpnI/HindIII, the purified target fragment was combined with the same cloning sites of the pCAMBIA1301 vector. The resulting plasmid was then transformed into Agrobacterium tumefaciens strain EHA105 and used to infect the cotyledons of the tomato ‘Ailsa Craig’ seedlings. Two independent Cas9-free T2 hsfA1a lines carrying mutations were identified for further experiments (HsfA1a (HsfA1a-OE2 and HsfA1a-OE5) used for the experiments were constructed as previously described [To obtain d method . The tareriments . Two indEight-week-old (flowering stage) tomato plants were transferred to the growth chamber at 39°C for 3 hours. Mature pollen grains were separated from anthers for viability and germination rate determination according to the methods described previously . The polhttp://www.lc-bio.com/). Poly(A) RNA was obtained using magnetic beads linked to poly-T oligonucleotides and then fragmented into small 300-bp (±50-bp) pieces and reverse-transcribed to generate cDNA. Sequencing was then conducted with an Illumina Hiseq 4000 according to the supplier’s recommended methods. Expression of all transcripts were estimated using StringTie [2(FPKM) | >1 and P <. 05.Total RNA was obtained and purified with TRIzol reagent from 0.5-g anther samples . The RNA-seq experiment was performed in accordance with the manufacturer’s protocol, and the data were analyzed by LC Biotech  staining was used to detect ROS accumulation in tomato anthers according to previously described methods [2•− in anthers was detected by NBT  staining according to previously described methods [https://imagej.nih.gov/ij/) as described previously [H methods . The O2•eviously .Anther tissue (0.3 g) was homogenized with 3 ml of cold enzyme buffer and centrifuged at 12 000 g for 10 minutes. The resulting supernatant was then used to measure enzymatic activity with a UV-2410PC spectrophotometer . The activities of SOD, POD, CAT, and ascorbate peroxidase were measured according to the previously described protocol . MDAR 4 and GST In order to detect autophagosomes in pollen, MDC staining was performed as previously described . The sam® 480 II Real-Time PCR detection system (Roche). Tomato Actin and SAND acted as internal controls, and the corresponding primer sequences used are shown in Total RNA of tomato anthers was obtained using the Easy Plant RNA Kit  and reversed to cDNA using the ReverTra Ace qPCR RT Kit . qRT–PCR used SYBR Green PCR Master Mix  and the LightCyclerhttps://solgenomics.net. The probes were biotin-labeled and annealed using a Biotin 30 End DNA Labeling Kit . EMSA experiments were conducted using the LightShift Chemiluminescence EMSA Kit . The probe sequences used in the experiment are shown in Recombinant protein preparation of tomato HsfA1a has been described . The 1.5The ChIP assay was performed with the EpiQuik™ Plant ChIP Kit . After 3 hours of heat stress treatment at 39°C, 1.5 g of anther tissues was obtained from HA-tagged HsfA1a-OE and WT plants and the input chromatin was extracted. For ChIP experiments, HA antibody  was used for chromatin immunoprecipitation, and anti-mouse IgG  was used as a negative control. The primers used in ChIP–qPCR analysis are specifically designed according to the target gene promoter, as shown in Total protein was extracted from 0.1-g tomato anther samples with 300 μl protein extraction buffer. SDS–PAGE of denatured proteins with a 13.5% gel containing 6 M urea was used to detect Atg8 . WesternP < .05.Five independent replicates for each determination were performed and statistical analysis was performed using SPSS statistical software . Experimental data were analyzed using Tukey’s test at This work was supported by the National Key Research and Development Program of China (2018YFD1000800), the National Natural Science Foundation of China (31922078 and 31872089), the Fundamental Research Funds for the Central Universities (226-2022-00122), and the Starry Night Science Fund of Zhejiang University Shanghai Institute for Advanced Study (SN-ZJU-SIAS-0011).D.-L.X. and J.Z. conceived this study and wrote the manuscript. D.-L.X., H.-M.H., C.-Y.Z., C.-X.L., M.K.K., and Z.-Y.Q. performed the experiments. D.-L.X. and J.Z. analyzed the data. All authors read and approved the manuscript.The data supporting the findings of this study are available from the corresponding author (J.Z.) upon request.No potential conflict of interest was reported by the authors.Web_Material_uhac163Click here for additional data file.


Theoretically, the CPEs with the same molecular structure usually display similar photoelectronic performances. However, the resulting PBTBTz-SO3Na reveal a similar light absorption property, but different luminescence. The higher molecular weight is, the stronger the fluorescence intensity of PBTBTz-SO3Na that occurs. PBTBTz-SO3Na with different molecular weights have different colors of luminescence. It is well known that the molecular aggregates often led to weaker luminescent properties for most of the conjugated polymers. However, PBTBTz-SO3Na exhibits a higher molecular weight with an increasing molecular chain aggregation, i.e., the nanoaggregates of PBTBTz-SO3Na are beneficial to emission luminescence. This work provides a new possible chemical design of CPEs with a controllable, variable luminescence for further optoelectronics and biomedicine applications.Due to their distinctive intrinsic advantages, the nanoaggregates of conjugated polyelectrolytes (CPEs) are fascinating and attractive for various luminescence applications. Generally, the emission luminescence of CPEs is determined by the conjugated backbone structure, i.e., different conjugated backbones of CPEs produce emission luminescence with different emission wavelength bands. Here, we polymerized the bis(boronic ester) of benzothiadiazole and an alkyl sulfonate sodium-substituted dibromobenzothiatriazole to provide PBTBTz-SO Conjugated polyelectrolytes (CPEs) with a conjugated backbone and a charged flexible side-chain are employed because of their strong light-harvesting ability, excellent water solubility and good photo-stability, etc. ,2,3,4,5.The powerful applications promote the design and synthesis of CPEs through the structural modifications of the side groups, the conjugated backbone, the ionic counterions and their functionalities ,17,18,19Developing innovative aggregated states are also another important consideration that aim to keep abreast with the development of the applied fields for CPEs . The aggBenzotriazole (BTz) and benzothiadiazole (BT) have been proven to be two of the most promising units for the D-A conjugated polymers which are used in efficient optoelectronic devices because of their strong electron affinity ,21,22,233Na) with a sulfonate sodium charged-end side chain, but they are based on the BTz and BT conjugated backbones with different molecular weights. The resulting PBTBTz-SO3Na reveal similar light absorption properties, but they have different fluorescence performances. By measuring their optoelectronic properties along with the morphology characteristics, which were determined by the UV-vis absorption, photoluminescence (PL) and transmission electron microscopy (TEM), etc., we can further investigate the relationship among the lengths of the conjugate backbone, the molecular aggregated states and the luminescence performances of PBTBTz-SO3Na with various molecular weights. As a result, the molecular weight of PBTBTz-SO3Na plays a critical role in the intrachain and interchain energy transfer, which result from the various aggregates and electrostatic stabilization by the charges on the backbone. Such results will be provide the information about the structure–property relationship, and further insights into the working mechanisms will be gained for promising luminescence applications of CPEs.Herein, we report CPEs  of benzothiadiazole and an alkyl sulfonate sodium-substituted dibromobenzothiatriazole yielded PBTBTz-SO3Na, which is soluble in highly polar solvents, such as water and methanol, etc. All of the synthetic preparations and representations are demonstrated in detail in 3Na with different molecular weights was achieved by controlling the ratio of the monomer and the catalyst as shown in the SI. The ion exchange step further generated PBTBTz-SO3TBA through the exchange procedure in The synthetic procedure and chemical structure of PBTBTz-SO3Na were maintained. The PBTBTz-SO3Na with larger molecular weights for P1, P2 and P3 were designed as per 3Na with different lengths of conjugated backbone are exhibited in To examine the role of molecular weight, the ion-charged end and backbone structure of PBTBTz-SO3Na appeared as the molecular weight increased [The number average molecular weights of P1, P2 and P3 were obtained, and these have values of 88 kDa, 94 kDa and 157 kDa, respectively, by GPC in ncreased . The opt3Na is more inclined to obtain a smaller bandgap. The electrochemical properties of P1, P2 and P3 were investigated by the cyclic voltammetry in 3Na increases, some small platelet-like molecular chains occur, as can be obviously seen in 3Na with a larger molecular weight, which is consistent with the trend represented by the GPC results.The molecule chains aggregates of the P1, P2 and P3 films were performed to further study the relationship between the molecular weight and morphology by TEM ,44,45,463Na. The photograph of the P1, P2 and P3 (0.016 mg/mL) solutions used for the PL measurement is displayed in 3Na with a higher molecular weight exhibits a stronger fluorescence intensity, implying that the increasing molecular chain aggregation of PBTBTz-SO3Na is beneficial to the luminescence. As seen from the analysis results of λEX, the higher the molecular weight is, the more ordered aggregations of PBTBTz-SO3Na occur. It is well known that interchain aggregation has led to the decreased fluorescence intensity for the most conjugated polymer [3Na solution illustrates the opposite trend; the nanoaggregates result in a stronger luminescent emission. The emission maxima of P1, P2 and P3 are located at 560 nm, 620 nm and 565 nm, respectively. The fluorescence photograph of P1, P2 and P3 in H2O at concentrations of 5 mg/mL is shown in 3Na with different molecular weights have different colors of luminescence. The fluorescence quantum yields of P1, P2 and P3 in H2O were determined relative to a reference sample with a known quantum yield. The reference sample here is rhodamine 101 inner salt in H2O. All of the PBTBTz-SO3Na solutions retains a strong fluorescence intensity, especially for the P2 and P3 solutions, and the fluorescence quantum yields of P1, P2 and P3 with the concentration of 0.016 mg/mL are 3.4%, 10% and 34%, respectively, as can be seen in The photoluminescence (PL) was further investigated to understand the relationship between the optical property and the molecular aggregated states of PBTBTz-SOnd P3 0.0 mg/mL so polymer ,18,19,203Na samples with different molecular weights were achieved by controlling the polarity of the solvent and the ratio of the monomer and the catalyst. The molecular weight of PBTBTz-SO3Na plays a critical role in the intrachain and interchain energy transfer and photoluminescence as a result of the differences in the electrostatic stabilization by the charged pendants on the backbone. With the lengthening of the conjugated backbone, the molecular chain maintains a highly ordered arrangement, and the twist of the molecular main chain shows a gradual increase. The PBTBTz-SO3Na with a larger molecular weight shows more absorption peaks, indicating more ordered microstructures of PBTBTz-SO3Na have appeared as the molecular weight increases. Meanwhile, the increasing platelet-like nanomorphology indicates a stronger aggregation of PBTBTz-SO3Na with the larger molecular weight, as seen from the TEM mages. The PL is further investigated to understand the relationship between the optical property and molecular aggregated states of PBTBTz-SO3Na. PBTBTz-SO3Na with different molecular weights have different colors of luminescence. All of the PBTBTz-SO3Na solutions retain a strong fluorescence intensity, especially for the P2 and P3 solutions, and the fluorescence quantum yields of P1, P2 and P3 with the concentration of 0.016 mg/mL are 3.4%, 10% and 34%, respectively. The higher the molecular weight is, the stronger the aggregation and luminescence that can occur for PBTBTz-SO3Na. The phenomenon may be attributed to the twist of the molecular main chain, which inhibits the photoinduced electron transfer among the conjugated backbone.PBTBTz-SO


Existing molecular property prediction methods based on deep learning ignore the generalization ability of the nonlinear representation of molecular features and the reasonable assignment of weights of molecular features, making it difficult to further improve the accuracy of molecular property prediction. To solve the above problems, an end-to-end double-head transformer neural network (DHTNN) is proposed in this paper for high-precision molecular property prediction. For the data distribution characteristics of the molecular dataset, DHTNN specially designs a new activation function, beaf, which can greatly improve the generalization ability of the nonlinear representation of molecular features. A residual network is introduced in the molecular encoding part to solve the gradient explosion problem and ensure that the model can converge quickly. The transformer based on double-head attention is used to extract molecular intrinsic detail features, and the weights are reasonably assigned for predicting molecular properties with high accuracy. Our model, which was tested on the MoleculeNet  benchmar Molecular property prediction refers to the effective identification of molecular properties such as lipophilicity, binding affinity, biological activity, and toxicity . For fieThe quantitative structure-activity/property relationship (QSAR/QSPR) has always been a hot topic in materials chemistry . This meWith the rise of artificial intelligence, combining artificial intelligence with the field of molecular property prediction has become a major research trend for improving the accuracy of molecular property prediction –14. CurrCommonly used prediction models are ridge regression, random forest(RF), elastic network, support vector machine(SVM), gradient boosting and extreme gradient boosting (XGBoost). Ridge regression  is a regOverall, machine learning methods require domain experts to extract features manually, but their handcrafted molecular descriptors are easily limited by the subjective experience and knowledge of the experts.Unlike machine learning methods, deep learning enables features to be extracted automatically, so deep learning methods are particularly suitable for molecular property prediction. The feed-forward neural network (FFN) is one of the simplest artificial neural network . The neuA new neural network framework, DHTNN, is proposed; it uses an activation function (Beaf), residual network, and transformer based on Double-head attention to process and extract molecular features for high-precision molecular property prediction.A new activation function, Beaf, is defined, which can nonlinearize the molecular characteristics. Compared with other activation functions, the performance of our model DHTNN using the activation function beaf is improved.The molecular residual network is introduced to solve the gradient problem of the neural network and ensure that the model can converge quickly.The Transformer based on Double-head attention extracts the intrinsic detailed features of molecules and acquires global information in parallel, effectively improving the performance of the model in predicting molecular properties.Our method was experimentally tested on six datasets from the MoleculeNet  benchmarIn conclusion, we found that the current molecular property prediction based on deep learning techniques has the problem of low prediction accuracy. The main reason for this problem is poor generalization ability due to the use of traditional activation functions, such as ReLU, PReLU, and Tanh, in the nonlinear representation of molecular features. There may be problems with gradient disappearance or explosion in the network. The global information cannot be taken into account when molecular detail features are extracted. In this regard, this paper makes the following contributions. The neural network framework is divided into three parts, as shown in Fig. In this paper, a DHTNN is proposed for molecular property prediction. The molecular residual network encoding structure is proposed in this neural network framework structure, in which a graph convolution neural network is used for the message passing process. Hence, for any molecular dataset, the input molecular chemical formula needs to be first converted into the form of a molecular map. In order to facilitate data reading and memory saving by computers, the molecular chemical formula is usually represented by a matrix , 47, 48,entclass1pt{minimaentclass1pt{minimax denotes the input, andTo address the shortcomings of the existing activation functions, such as Tanh is easy to saturate, the negative part of ReLU cannot be mapped, and the negative part of ELU and GeLU are not mapped accurately. In this paper, we propose the activation function Beaf, which is more suitable for molecular feature nonlinearization mapping and has better generalization. The specific equation is as follows:entclass1pt{minimaentclass1pt{minimaentclass1pt{minimaentclass1pt{minimaentclass1pt{minimaAfter the high-precision nonlinear generalization representation of the molecular features in "This acts on the molecular map for encoding. The directed MPNN can be divided into two phases: the directed message passing phase and the readout phase.The directed MPNN needs to initialize the hidden state of the bond ..\documenquations  and 8)..\documenWhen training a neural network, the parameters of the previous layer affect the input of the later layer, thus making the training complicated. This requires normalizing the encoded data, adjusting the distribution of the data, reducing the internal covariance bias, and improving the training speed. Therefore, batch normalization is required to optimize the mean position and variance size to make the new data distribution more closely match the real data distribution.Normalization is done mainly by processing the mean  and four classification datasets . The algorithm evaluation metric for the regression dataset was the root mean square error (RMSE), which is the arithmetic square root of the expected value of the squared difference between the parameter estimate and the true value of the parameter. A smaller RMSE indicates a smaller error and better prediction performance. The algorithm evaluation metrics for classification datasets were the area under the recall curve (PRC-AUC) and the area under the receiver operating characteristic curve (ROC-AUC) . Larger In order to verify the algorithmic effectiveness of our proposed activation function Beaf on our model, we performed validation experiments on the activation function selection. On the six datasets , we applied the activation functions Beaf, ELU and GeLU to our algorithmic model and compared their performances, shown in Tables The Lipophilicity and PDBbind datasets, shown in Table In Table In conclusion, for ELU, all experimental results based on the Beaf are better than those based on the ELU on the datasets Lipophilicity and PDBbind. For GeLU, on the four datasets , only on the BACE dataset, the experimental results based on the GeLU are slightly better than those based on the Beaf. The experimental results of the algorithmic model based on the Beaf are better than those of the algorithmic model based on the GeLU on three of the four datasets. Therefore, we chose Beaf as the activation function for the double-head transformer neural network (DHTNN) for molecular property prediction.Our experiments were run on a Windows 10 operating system with a 1.70 GHz Intel Xeon Bronze 3104 CPU, 64 GB of RAM, and an NVIDIA RTX2080 GPU, using python 3.8 as the development language and PyTorch 1.5.1 as the neural network framework for deep learning training.The results of our algorithm were compared with the following state-of-the-art methods: MolNet , RF on MThe methods used for performance comparison included machine learning methods and deep learning methods, and RF on Morgan is currently the most advanced method for machine learning. MolNet, FFN on Morgan, FFN on Morgan Counts, FFN on RDKit and DMPNN are current advanced methods for deep learning.For the regression dataset, we calculated the RMSE to evaluate the performance of the algorithm. The lower the RMSE, the better the model performance. As shown in Figs. ng Table . Our modFor the classification dataset, we calculated the PRC-AUC and ROC-AUC. The higher the AUC, the better the model performance. As shown in Figs. Whether on regression or classification datasets, our model did not exhibit gradient disappearance or explosion. The molecular residual network encoding in the model played an important role in ensuring that the model converged.In this paper, a new algorithmic framework, DHTNN, was proposed for molecular property prediction. Beaf, a new activation function, is included in the molecular nonlinear representation part, and the negative part is also able to be mapped, making the mapping more accurate and improving the model nonlinear representation accuracy and its generalization ability. In the molecular encoding part, the addition of the residual network prevents the gradient from disappearing or exploding and ensures that the model can converge. In the extraction of molecular features, the involvement of the Transformer based on Double-head attention can focus on the features of the region of interest for the prediction results and assign the weights reasonably. Running our model on six datasets, our method outperformed current state-of-the-art methods in all metrics. The experimental results demonstrate the effectiveness of our proposed algorithmic framework.


Coronary computed tomography angiography (CCTA) represents a common approach to the diagnostic workup of patients with suspected coronary artery disease. Technological development has recently allowed the integration of conventional CCTA information with spectral data. Spectral CCTA used in clinical routine may allow for improving CCTA diagnostic performance by measuring myocardial iodine distribution as a marker of first-pass perfusion, thus providing additional functional information about coronary artery disease. The diagnostic performance of coronary computed tomography angiography may be expanded by using spectral data.Iodine perfusion maps can functionally assess the impact of coronary stenosis.Myocardial iodine distribution may be used as a marker of myocardial perfusion.The use of coronary computed tomography angiography (CCTA) has seen a major increase in the last decade as a first-line noninvasive technique for evaluation of coronary artery disease, in patients with low or moderate risk of disease. Nowadays, CCTA is able to detect coronary stenoses with excellent sensitivity and negative predictive value . AdditioTechnological advances have furtherly broadened the potential of this technique for cardiovascular applications by the introduction of spectral imaging , 4. In pThe main approaches used to obtain dual-energy imaging are dual-source platforms, made by two x-ray tubes and detectors that simultaneously acquire data; voltage-switching platforms, where a single x-ray tube rapidly switches between low- and high-energy levels; and dual-layer spectral detector platforms, which allow to collect low-energy and high-energy data at the same time with a single detector, composed by two scintillation layers. The sensitivity of each layer is determined by its composition: low-energy x-rays are detected by an yttrium-based top layer, whereas a gadolinium-oxysulfide bottom layer is sensitive to high-energy x-rays, providing a spectral dataset .At our centre, we have been performing CCTA on a dual-layer DECT system  for almost a year. Based on our experience, dual-layer dual-energy computed tomography (DECT) has proved to be a robust technique to assess first-pass myocardial iodine distribution during CCTA, by measurement of iodine maps on a per-segment basis . In factWe measured noninvasive cFFR based on our spectral CCTA datasets with dedicated software . Before the execution of CCTA, patients were medicated with a sublingual vasodilator agent  and patients whose heart rate was greater than or equal to 70 beats per minute were administered β-receptor blocker . The iodine concentration of all myocardial segments was measured by vendor-recommended software , and the segment with the least value was reported and assigned to left anterior descending artery (LAD), left circumflex (LCx), and right coronary artery (RCA), according to the standardised myocardial segmentation .i.e., ≤ 0.75) show a difference in iodine distribution between normally perfused and ischemic myocardial segments . On the other hand, no difference in terms of myocardial attenuation (HU) was observed .As a sample case, we show patient 1 (see Table ved Fig. . ConventOur initial experience indicates that iodine distribution maps may provide an additional functional parameter to assess coronary artery disease, which may further increase CCTA diagnostic performance.To the best of our knowledge, there are limited studies in the scientific literature that have evaluated the feasibility of myocardial perfusion imaging by means of dual-energy CT iodine distribution, and only a few have assessed the potential of spectral-detector platforms, using FFR as the reference standard –14.versus nonischemic segments, due to the limited sample size. Additional prospective multicentre studies with larger patient cohorts are needed to establish whether iodine distribution maps obtained from spectral datasets can be used as a surrogate marker of myocardial first-pass perfusion.This initial data does not currently allow for defining a cutoff value to distinguish between ischemic We hypothesise that iodine perfusion maps can help distinguish between hemodynamically significant and nonsignificant coronary stenoses, improving the diagnostic performance of CCTA.


However, most MOFs are insulative and their three-dimensional (3D) porous structures hinder the electron transfer pathway between the current collector and the redox enzyme molecules. In order to facilitate electron transfer, here we adopt two-dimensional (2D) metal–organic layers (MOLs) to support the HRP molecules in the detection of H2O2. The correlation between the current response and the H2O2 concentration presents a linear range from 7.5 μM to 1500 μM with a detection limit of 0.87 μM (S/N = 3). The sensitivity, reproducibility, and stability of the enzyme sensor are promoted due to the facilitated electron transfer.Metal–organic frames (MOFs) have recently been used to support redox enzymes for highly sensitive and selective chemical sensors for small biomolecules such as oxygen (O Employi[−1·cm−2 . With a  for HRP .2-Hf-BTB-MOL, as the supporter of HRP molecules to construct the electrochemical H2O2 sensor. The results show that the 2D MOL-based sensor cannot only facilitate the electron transfer by shortening the transfer distance, but also improve the effective loading amount of HRP, demonstrating the prospective applications of 2D MOLs in electrochemical enzyme sensors.A two-dimensional metal–organic framework has an ultrathin layer structure, which increases the exposed active sites, reduces the resistance and, ultimately, improves its catalytic performance. It is widely used in water splitting reactions, the electro-oxidation of organic molecules, etc. . Here, w2-Hf-BTB-MOL/MWNTs/CC (Curve c), and HRP/NH2-Hf-BTB-MOL/MWNTs/CC (Curve d) electrodes, are shown in 2-Hf-BTB-MOL/MWNTs/CC electrode was larger than that of the MWNTs/CC electrodes. Meanwhile, a pair of redox peaks at −0.38 V were observed in both Curve b and Curve d. It was observed that the Faraday current response on the HRP/NH2-Hf-BTB-MOL/MWNTs/CC electrode was enhanced much higher than that on HRP/MWNTs/CC electrode. The ratio of Faraday current caused by the redox reaction of HRP over the charging/discharging current of EDL was also improved on the HRP/NH2-Hf-BTB-MOL/MWNTs/CC electrode. The results show the facilitated charge transfer between the MWNT/CC network and the HRP molecules immobilized on the 2D MOL through the static interaction between the protonated amino groups and HRP molecules.The voltammetric behaviors of the CMEs, including the MWNTs/CC (Curve a), HRP/MWNTs/CC (Curve b), NH2-Hf-BTB-MOL/MWNTs/CC electrode. Since the HRP molecules were immobilized on the 2D MOL, the typical voltammetric characteristics of redox adsorption species are presented. The peak potential hardly moved with the scanning rate, and the peak current was found in harmonious proportion to the scan rate , F is the Faraday constant , R is the gas constant (8.314 J·K−1·mol−1), and T is the experimental temperature (298.15 K), A is an apparent electrode area, Γ is the apparent surface concentration, and v is the scan rate (V·s−1). From Equation (1), Γ was calculated as 3.88 × 10−10 mol·cm−2 on the HRP/NH2-Hf-BTB-MOL/MWNTs/CC electrode, which was 3.3-fold higher than that on HRP/MWNTs/CC electrode (1.17 × 10−10 mol·cm−2), and 10-fold higher than that in previous reports [2O2 obtained on the HRP/NH2-Hf-BTB-MOL/MWNTs/CC electrode is shown in can rate C, indica reports ,22,23. TTo further elucidate the effect of the 2D MOL on the HRP redox reaction, EIS experiments were performed and the results are shown in 2-Hf-BTB-MOL/MWNTs/CC electrode in the absence of H2O2. The peak potential of HRP redox reduction shifted negatively with the increased pH value with a slope of −53.26 mV·pH−1, indicating it was a proton-coupled single electron transfer reaction [2-Hf-BTB-MOL at 0.25 mg/mL and the volume at 1 mL. From reaction . Conside2-Hf-BTB-MOL/MWNTs/CC electrode. The applied potential was fixed at −0.4 V and the steady-state Faraday current responses with the injected H2O2 concentration were recorded, as shown in 2O2 concentration ranged from 7.5 μM to 1.5 mM (−1 ·cm−2 and 0.87 μM (S/N = 3). The affinity of H2O2 on the HRP/NH2-Hf-BTB-MOL/MWNTs/CC electrode was also evaluated by the Lineweaver–Burk equation [mapp is the Michaelis–Menten constant, Iss is the steady-state current after H2O2 injection, C is the concentration of H2O2, and Imax is the limiting enzyme-catalytic current when H2O2 is saturated. The Kmapp was obtained as 0.22 mM from the linear relationship between 1/Iss and 1/C 1Iss2O2 (1 mM) in the experiment. As shown in 2O2 because of the specific reduction of HRP to H2O2. Moreover, five groups of parallel experiments were performed in the presence of 0.5 mM H2O2 and the relative standard deviation of the Faraday current was 1.36%, indicating the good reproducibility of the preparation protocols of the HRP/NH2-Hf-BTB-MOL/MWNTs/CC electrode. Moreover, after 50 cycles, the Faraday current was kept at 91.49% of the original  was purchased from Linde Gas Co.Carbon cloth (CC) was purchased from Carbon Energy Technology Co., Ltd. . Multi-walled carbon nanotubes (MWNTs) was purchased from Nanjing XFNANO Materials Technology Co., Ltd. . Nafion (5 wt%) was purchased from Guangzhou Lige Technology Co., Ltd. . Ready-to-use PBS powder was purchased from Shanghai Sangon Biotech Co., Ltd. . Horseradish peroxidase (HRP), uric acid (UA), ascorbic acid (AA), glucose (Glu), dopamine (DA), and tyrosine (Tyr) were obtained from Aladdin Biochemical Technology Co., Ltd. . Aqueous solutions used in the experiments were prepared with deionized water . Nitrogen gas (N2-Hf-BTB-MOL was prepared by grafting an amino-terminated group by stirring 30 mL Hf-BTB-MOL (60 mM based on Hf in H2O) and 50 mg ethanolamine phosphate at 60 °C overnight. HRP molecules were pre-loaded on the newly prepared NH2-Hf-BTB-MOL by ultrasonic dispersion  of their mixture with different mass ratios in a 400 μL PBS buffer , and a subsequent constant-temperature oscillation  for 1 h at 4 °C.The 2D MOL was a gift from Dr. Huihui Hu who is with Prof. Cheng Wang’ group at Xiamen University . NH2-Hf-2-Hf-BTB-MOL suspension was drop-coated on the MWNTs/CC and dried at room temperature. Finally, 20 μL Nafion solution (5 wt%) was dropped to solidify the coatings. The prepared HRP/NH2-Hf-BTB-MOL/MWNTs/CC electrode was stored in a refrigerator at 4 °C for use. For comparison, the MWNTs/CC, HRP/MWNTs/CC, and NH2-Hf-BTB-MOL/MWNTs/CC electrodes were also prepared with the same protocols as schemed in 2-Hf-BTB-MOL/MWNTs/CC-modified electrodes are shown in Carbon cloth (CC) was cut into pieces of 1 cm × 1 cm, alternately washed with acetone, ethanol, and water for 5 min, and dried on a heating plate. Then, 1 mg/mL MWNTs was dispersed ultrasonically in ethanol solution for 30 min. Subsequently, a 20 μL MWNTs suspension was dropped onto the CC piece to form a MWNTs/CC current collection network. After that, 20 μL HRP/NH2 to degas oxygen before experiment.Electrochemical experiments were performed with a CHI 850D electrochemical workstation . Electrochemical impedance spectroscopy (EIS) experiments were performed with Autolab electrochemical workstation . A three-electrode system was adopted, where a saturated calomel electrode (SCE) and a platinum (Pt) wire electrode served as the reference electrode and counter electrode, respectively. The solution was bubbled gently by high-purity N2-Hf-BTB-MOL material as the support for HRP, we developed an electrochemical biosensor for the rapid, reliable, and precise detection of H2O2. Both the direct redox behaviors of HRP molecules and the electrocatalytic reduction of H2O2 were observed on the HRP/NH2-Hf-BTB-MOL/MWNTs/CC electrode. By optimizing the pH value and effective loading amount of HRP, we found that the electrochemical enzyme sensor has excellent sensitivity, selectivity, affinity, and stability to H2O2. All the results indicate that the 2D MOLs have significant potential in the applications of electrochemical enzyme sensors because the ultrathin 2D MOLs cannot only facilitate the electron transfer, but also improve the effective loading amount of the enzyme molecules.Employing a 2D NH


Escherichia coli and Klebsiella pneumoniae isolates. The observed inverse relationships were confirmed by linear regression analysis. Similar results were obtained for the vaccination coverage in the overall population. Despite the main limitations of our study, its findings suggested an inverse relationship between influenza vaccination coverage and AMR proportions over the years, which was in accordance with previous theories illustrating how influenza vaccination reduced antibiotic prescriptions. However, since our study did not allow for us to elucidate the mechanisms underpinning the observed relationship, more and better data on confounding and mediating factors should be considered in future research. Antimicrobial resistance (AMR) is one of the key threats to global health and requires sustainable and effective actions to reduce its consequences in the near future worldwide. There are several well-documented and epidemiologically supported examples that both bacterial and viral vaccines might have an impact on AMR. Here, we conducted an ecological analysis of Italian data to evaluate the relationship between influenza vaccination coverage and AMR proportions over the last two decades. We found significant negative correlations between influenza vaccination coverage in the population over 64 years and AMR in  Klebsiella pneumoniae resistant to third-generation cephalosporins and carbapenems have been reported in several countries [Acinetobacter spp. that are resistant to carbapenems are of concern [Antimicrobial resistance (AMR) is one of the most important challenges that modern medicine must face. Each year, there are over 670,000 infections due to antimicrobial-resistant bacteria in the European Union (EU/EEA) and approximately 33,000 deaths as a direct result of these infections . It has Thus, robust investments to tackle AMR are urgently necessary and would have a significant positive effect on healthcare systems, as well as on population health in general. For these reasons, efforts to prevent and control AMR are essential for the Public Health agenda. As AMR is among the ten key threats to global health , the heaDrivers that support mechanisms for the spread of AMR include the growing and often inappropriate use of antimicrobials, in both the medical and veterinary fields. One example is the use of antimicrobials, either prophylactically or therapeutically, for viral infections . The traIn this complex scenario, there are several well-documented and epidemiologically supported examples of both bacterial and viral vaccines having an impact on AMR ,17. In pVaccination is certainly the most effective measure against influenza, and, at the European level, the strategy is generally to protect people at higher risk of complications if infected. Accordingly, the WHO recommends influenza vaccines for pregnant women, children aged 6–59 months, the elderly, healthcare workers, and individuals with chronic medical conditions. In 2003, the World Health Assembly offered its support to reach the auspicial target of 75% vaccination coverage in older age groups by 2014–2015, a coverage level settled by the Health Council of all Health Ministers. However, despite several efforts, influenza vaccination coverage is lower than this target in almost all European countries . In factK. pneumoniae and A. baumannii [Given this, our hypothesis was that influenza vaccines might at least partially mitigate the spread of AMR pathogens at international and national levels. To explore this hypothesis, in the current study, we focused on Italy, one of the Member States with the highest level of AMR in Europe . This isaumannii . For allWe first collected national data on influenza vaccination coverage in the overall population and among those aged more than 64 years. These data were released by the Italian National Institute of Health   and madeA. baumannii resistant to aminoglycosides, carbapenems, or fluoroquinolones;Escherichia coli resistant to fluoroquinolones, third-generation cephalosporins, aminoglycosides, or aminopenicillins;K. pneumoniae resistant to Carbapenems, third-generation Cephalosporins, Aminoglycosides, or Fluoroquinolones;Staphylococcus aureus resistant to Methicillin (MRSA);Pseudomonas aeruginosa resistant to Fluoroquinolones, Piperacillin and Tazobactam, Carbapenems, or Ceftazidime;Streptococcus pneumoniae resistant to Penicillins or Macrolides.We also collected data on AMR percentages for selected isolates and specific periods in Italy, when available. AMR percentages on the total of tested isolates were provided by the Italian AMR surveillance project of the ISS (AR-ISS), which was based on a network of sentinel laboratories reporting data on the antimicrobial susceptibility of bloodstream isolates of selected species . The AR-Additionally, data on antimicrobial use in the general population were extracted from the last National Report on Antibiotic use in Italy, published by the Italian Medicines Agency through the Medicines Utilisation Monitoring Centre in 2020 . AvailabTo consider the potential effect of socio-demographic indicators, data on the annual number of Italian residents, proportion of men, and aging index were provided and obtained by the Italian National Institute of Statistics  . In partSummary statistics were used to describe influenza vaccination coverage, AMR percentages and other collected measures, using mean, standard deviation (SD), and range, when necessary. First, we tested for bivariate correlations between influenza vaccination coverage and AMR percentages, using the Spearman’s rank correlation coefficient (rho). We also tested for cross-correlations between the time-series with a maximum lag/lead time of 3 years. For combinations with statistically significant correlations, we plotted the annual influenza vaccination coverage against AMR percentages through overlaid scatter plots. To consider potential confounding factors, we also evaluated the correlations between influenza vaccination coverage and AMR proportions and the number of tested isolates, data on antibiotic use, and socio-demographic indicators. Finally, we performed linear regression analyses, using influenza vaccination coverage as the independent variable and percentage of AMR as the dependent variable. For each combination, the analyses were adjusted for the annual number of tested isolates and the aging index. The results were reported as β and its standard error (SE). All the analyses were performed on the SPSS software , with a significance level α of 0.05.E. coli, 34,455 for K. pneumoniae, 8868 for A. baumannii, 58,468 for S. aureus, 6014 for S. pneumoniae, and 17,963 for P. aeruginosa. Notably, the annual number of tested isolates increased over the years. The highest average percentages of AMR were reported for A. baumannii, independent of the considered antimicrobial. By contrast, the average percentages of AMR varied for E. coli: from 15.1% for aminoglycosides to 61.9% for aminopenicillins. A wide variation was also observed for K. pneumoniae: from 21.7% for carbapenems to 46.7% for third-generation cephalosporins. The average percentage of S. aureus resistant to methicillin was 36.5%, while lower percentages were reported for P. aeruginosa and S. pneumoniae, respectively. Regarding antibiotic use in the general population, the average value was of 16.9 DDD per 1000 persons each day. The average antibiotic use referred to the period from 2013 to 2019, and its annual value slightly decreased from 18.4 in 2013 to 15.6 in 2019. E. coli and K. pneumoniae . Regarding E. coli, the greatest correlation coefficient was observed for resistance to fluoroquinolones, followed by third-generation cephalosporins, aminoglycosides, and aminopenicillins. Cross-correlation analysis demonstrated that the highest correlation coefficients were obtained without a time lag between the time-series. The only exception was for E. coli that were resistant to aminoglycosides, which showed the highest correlation coefficient at a 2-year lag. Regarding K. pneumoniae, the greatest correlation coefficient was observed for fluoroquinolones, followed by third-generation cephalosporins, carbapenems, and aminoglycosides. Cross-correlation analysis demonstrated that the highest correlation coefficients were obtained without a time lag between the time-series. A negative correlation was also observed for P. aeruginosa that were resistant to piperacillin and tazobactam (p = 0.001), while no significant correlations were evident for the remaining combinations of isolates and antimicrobials. Similar results were obtained when considering influenza vaccination coverage in the over-64 population. In particular, vaccination coverage negatively correlated with percentages of AMR of E. coli and K. pneumoniae . Regarding E. coli, the greatest correlation coefficient was observed for resistance to fluoroquinolones, followed by aminoglycosides, third-generation cephalosporins, and aminopenicillins. Regarding K. pneumoniae, the greatest correlation coefficient was observed for aminoglycosides, followed by fluoroquinolones, carbapenems, and third-generation cephalosporins. A negative correlation was also observed for P. aeruginosa that were resistant to piperacillin and tazobactam (p = 0.001), while no significant correlations were evident for the remaining combinations of isolates and antimicrobials.p-values > 0.1). With respect to the aging index, we observed a negative but non-significant correlation . Similarly, vaccination coverage did not correlate with antibiotic use (p = 0.589), although this analysis was limited to the 2013–2019 period. Finally, we found negative correlations with the number of K. pneumoniae and P. aeruginosa tested , but not with other isolates . No correlations were evident between vaccination coverage in the overall population and the abovementioned measures and indicators. To test whether other measures and indicators might confound the observed relationships, we evaluated potential correlations of influenza vaccination coverage with socio-demographic indicators, data on antibiotic use, and number isolates tested each year . NotablyE. coli resistant to fluoroquinolones, third-generation cephalosporins, aminoglycosides, and aminopenicillins; K. pneumoniae resistant to fluoroquinolones, third-generation cephalosporins, carbapenems, and aminoglycosides; P. aeruginosa resistant to piperacillin and tazobactam) . By contrast, the aging index showed positive correlations with the proportion of E. coli resistant to third generation cephalosporins  and aminopenicillins , with the proportion of K. pneumoniae resistant to fluoroquinolones , third-generation cephalosporins  and carbapenems , and with P. aeruginosa resistant to piperacillin and tazobactam . Moreover, we observed positive correlations between AMR proportions and the number of isolates tested for all microorganisms under investigation . We also tested for correlations between AMR proportions, socio-demographic indicators, data on antibiotic use, and the number of isolates tested each year. The analysis was limited to specific combination of isolates and antimicrobials exhibiting significant correlations with vaccination coverage  . NotablyE. coli and K. pneumoniae. E. coli resistant to fluoroquinolones, third-generation cephalosporins, aminoglycosides, and aminopenicillins. After adjusting for the number of tested isolates and for the aging index, the analysis confirmed significant and negative relationships with percentages of resistance to third-generation cephalosporins , fluoroquinolones , aminoglycosides , and aminopenicillins . Next, we tested the linear relationship between influenza vaccination coverage in population over 64 years and AMR of K. pneumoniae resistant to carbapenems, third-generation cephalosporins, aminoglycosides, and fluoroquinolones. After adjusting for the number of tested isolates and for the aging index, the analysis confirmed significant and negative relationships with percentages of resistance to carbapenems , fluoroquinolones , and third-generation cephalosporins , but not with aminoglycosides . A negative relationship was also evident for P. aeruginosa that were resistant to piperacillin and tazobactam .Similarly, E. coli and K. pneumoniae. Regarding E. coli, the analysis confirmed a significant and negative relationship with the percentages of resistance to aminoglycosides , third-generation cephalosporins , fluoroquinolones , and aminopenicillins , after adjusting for covariates. We also tested the linear relationship between influenza vaccination coverage in the overall population and AMR of K. pneumoniae, the analysis confirmed significant and negative relationships with the percentages of resistance to fluoroquinolones  and third-generation cephalosporins , after adjusting for covariates. By contrast, no significant relationship was evident for resistance to aminoglycosides  and carbapenems .Regarding To our knowledge, the present study is the first to evaluate the relationship between influenza vaccination coverage and AMR at population level. To do that, we performed an ecological analysis of Italian data, which revealed an inverse correlation between influenza vaccination coverage and AMR for selected microorganisms and antimicrobials. K. pneumoniae and P. aeruginosa). Antibiotic use in the general population, instead, did not correlate with influenza vaccination coverage. Based on these results, we also performed linear regression analyses by adjusting for the aging index and for the number of isolates that were tested annually. In particular, influenza vaccination coverage in the population over 64 years exhibited an inverse relationship with the proportions of AMR in E. coli and K. pneumoniae. For E. coli, inverse relationships were evident for the percentages of resistance to third-generation cephalosporins, fluoroquinolones, aminoglycosides, and aminopenicillins. For K. pneumoniae, inverse relationships were evident for the percentages of resistance to carbapenems, fluoroquinolones, and third-generation cephalosporins. A negative relationship was also evident for P. aeruginosa that was resistant to piperacillin and tazobactam. Similar results were obtained considering vaccination coverage in the overall population. These findings, however, should be interpreted with caution due to the potential effect of unmeasured and non-available factors that might influence and/or mediate the observed relationship. In fact, several other factors contribute to AMR besides influenza vaccination, such as socio-demographic and patient’s characteristics, health conditions, vaccine type and supply, local public health condition, medication, incidence of infections, and antibiotic use. In our study, we also evaluated the relationships of influenza vaccination coverage with some socio-demographic indicators , the number of isolates tested annually, and antibiotic use in the general population. Accordingly, we noted an inverse but non-significant correlation with the aging index of the Italian population, and a significant inverse correlation with the number of tested isolates  Work Stream II Team conducted a mixed-methods study, which included a literature review of research articles, public health reports, and national action plans against AMR and ad-hoc interviews with experts on influenza and AMR . RegardiAs stated above, our results should be interpreted cautiously due to the ecological nature of our analysis  and the Data coverage was one of the main limitations of our study. Data related to vaccination coverage indicate the number of vaccine doses administered over the overall population or residents aged over 64 years. However, certain inaccuracies cannot be completely excluded, mainly due to the decentralized Italian Health System and to the different characteristics and functionalities of immunization registries implemented at regional levels . DespiteIn conclusion, the present study proposed an inverse relationship between influenza vaccination coverage and AMR. Our results were in accordance with previous theories illustrating how influenza vaccination reduced prescriptions for secondary bacterial infections, as well as the inappropriate antimicrobial use against acute febrile illnesses. However, our study design and results did not allow us to demonstrate this effect. For this reason, more and better data on confounding and mediating factors should be available to confirm our findings and to elucidate the mechanisms underpinning this relationship. The positive outlook is that future vaccines targeting other respiratory viruses could result in an even more marked reduction in antimicrobial use and AMR. For these reasons, further research should be encouraged to better evaluate the effectiveness of viral vaccination programs in impacting AMR.


IGF2, HMGA2, PLAG1, CDKN1C have also been identified in patients with SRS phenotypes. However, routine molecular diagnostics usually focus on 11p15 and chromosome 7, while less frequent causes are not systematically addressed.Silver-Russell syndrome (SRS) is a genetic disorder characterized by intrauterine and postnatal growth restriction, relative macrocephaly at birth, body asymmetry and typical facial features. Clinical and molecular heterogeneity is described in SRS. Common causes are loss of methylation of the imprinting center 1 in 11p15 and maternal uniparental disomy of chromosome 7. Other genetic alterations include disturbances of imprinted regions in 14q32, 7q32 and 11p15 as well as submicroscopic deletions and duplications. Single nucleotide variants in genes like PLAG1 as the only protein-encoding gene with known function.Here we report two patients with SRS features in which molecular karyotyping revealed microdeletions in 1q21 and 8q12.1 respectively. In a 3.5-year-old girl with postnatal growth restriction, feeding difficulties, relative macrocephaly and distinct SRS features a 2 Mb deletion in 1q21.1q21.2 was identified. Our second case is a 1.5-year-old boy with intrauterine and postnatal growth restriction, feeding difficulties and distinct facial features with a 77 kb deletion in 8q12.1 affecting PLAGL1 alteration is the smallest deletion in 8q12.1 ever reported in a patient with SRS phenotype and it finally confirms that PLAG1 is the SRS causing gene in 8q12.1. To increase the diagnostic yield in patients with suspected SRS, we recommend both molecular karyotyping and next generation sequencing-based approaches.The 1q21 region has not yet been assigned as an SRS region, although six patients with the same deletion and SRS features including relative macrocephaly have been described before. This new case adds to the evidence that distal 1q21 should be annotated as an SRS candidate region. The  Silver-Russell syndrome (SRS) is a congenital disorder, mainly characterized by severe intrauterine and postnatal growth restriction, relative macrocephaly at birth, body asymmetry and a typical facial gestalt (for review: ). Due toIGF2, HMGA2, PLAG1, CDKN1C), or with differential diagnoses of SRS  loss of methylation (LOM) in imprinting center 1 , (ii) maternal uniparental disomy of chromosome 7 (upd). AdditiRS e.g. –6)..IGF2, HMWhereas procedures to uncover molecular alterations affecting 11p15 and chromosome 7 are already implemented in routine molecular diagnostics of SRS , 5, 7.In addition to the major molecular SRS subgroups in 11p15, chromosome 7 and 14q32, genomic alterations within 1q21 and 8q12.1 have recently come into focus as SRS causing regions . These patients have either been identified by NGS-based approaches   (. In factencing)  and length  and a head circumference of 32 cm (z − 0.91).During pregnancy, the mother suffered from gestational diabetes, which was treated with modified diet. Intrauterine growth retardation (IUGR) was not reported, however, fetal hydronephrosis was diagnosed prenatally. Postnatally, vesicoureteral reflux led to recurrent episodes of pyelonephritis eventually making a reimplantation of both ureters necessary. In the postnatal period, the patient experienced feeding difficulties and a consecutive failure to thrive. Additional sip feed nutrition was started at the age of 2 years. The motoric and linguistic developmental milestones were reached within the normal range of time.At the age of 25 months, growth was restricted  and a further slowdown was observed at the age of 3.5 years . At that age, relative macrocephaly was reported . The patient exhibited a triangular face, asymmetrical palpebral fissures, epicanthal fold on the left side, short philtrum . The older brother was healthy and of normal height; a younger brother, born in the year of presentation of the patient, thrived well.By first line molecular SRS testing using methylation-specific multiplex ligation probe dependent amplification (MS MLPA) the characteristic molecular alterations on chromosomes 11p15, 7 and 14q32 were excluded. Molecular karyotyping  revealed heterozygosity for a 2 Mb deletion in 1q21.1q21.2  × 1) . One miscarriage in the 10th week of gestation has been experienced. The pregnancy resulted from spontaneous conception and was complicated by maternal cholestasis, GBS positivity (Group B Streptococcus infection) and IUGR. Abnormal color Doppler flowmetry prompted an urgent Caesarean section at 30 weeks of gestation. Birth weight, height and OFC were reduced (990 g (z − 1.79), 37 cm (z − 1.39), 27 cm (z -− .23)). Apgar scores were 8 and 9, respectively; the newborn required non-invasive ventilation in the first 4 days of his life. At the age of 10 days, his condition worsened due to sepsis, for which he required mechanical ventilation for three days, one unit of blood transfusion and eight days of combined antibiotic therapy. Echocardiogram and cranial US were normal. Hearing and vision are reported normal, development was in the normal range. At the age of 1 7/12 years growth was restricted , weight was 7.1 kg (z -3.87). Microcephaly was documented as well . The patient exhibited a triangular face and a protruding forehead  × 1); de-novo occurrence was proven by quantitative PCR of the parental DNA samples. In comparison to the other described cases carrying PLAG1 deletions, this patient exhibited the smallest deletion reported up to now mutations on chromosomes 11p15, 7 and 14q32 by MS MLPA was negative. By molecular karyotyping (CytoScan™ HD Array), a 77 kb deletion affecting the IGF2, PLAG1, and HMGA2 IGF2: SRS3, #616,489), 7p13-q32 , 8q12.1 , and 12q14 .Accordingly, the contribution of different genomic loci to the SRS phenotype is reflected by the suggestion of five different OMIM entries: chromosome 11p15.5  fulfilled the NH-CSS clinical score, but it should be noted that relative macrocephaly as an SRS key feature with its current definition (head circumference ≥ 1.5 SD above birth weight and/or length) could only be documented in two out of five patients. This case finally confirms that PLAG1 is the SRS causing gene in 8q12.1 and it can be assumed that the first deletion in this region reported in SRS in 1994 [The OMIM entry #618,907 for 8q12.1 is based on patients with single base pair substitutions in  review: ), but me in 1994  compriseBoth here discussed molecular subcohorts of patients with SRS features illustrate the clinical heterogeneity of SRS and the difficulty to establish the clinical diagnosis of SRS based on the current definition of the NH-CSS. For a more precise clinical diagnosis, the application of Human Phenotype Ontology (HPO) terms has to be discussed. Particularly with respect to the definition of the feature “relative macrocephaly” an expansion of the range needs to be considered. Furthermore, in daily clinical diagnostic routine, numerous patients are diagnosed as SRS due to their growth pattern and facial appearance, but do not pass the NH-CSS scoring as SRS. Since not even all of the patients with the typical SRS changes in 11p15 always fulfil the clinical score, it should be discussed to use the term SRS spectrum to reflect the clinical heterogeneity of the disorder.In both cases reported here, the molecular diagnoses were established by array-based molecular karyotyping. This confirms that CNV analysis is an essential tool in the diagnostic workup of patients with SRS features and has to be included in the diagnostic algorithm .GH1 gene: In our diagnostic cohort of patients with growth retardation, we recently missed a homozygous GH1 deletion by molecular karyotyping with the CytoScan™ HD Array as this assay does not cover this gene. However, we identified the alteration by WES (unpublished data). This example, as well as the broad pattern of molecular alterations identified in patients with SRS features, highlight the requirement of applying comprehensive assays, starting with first tier tests targeting aberrant methylation at different imprinted loci , followed by WES and WGS addressing both single nucleotide variants as well as CNVs [In the future, it will be interesting to see whether the increasing implementation of improved diagnostic NGS-based approaches will replace the separate CNV analysis by array analysis as WES- or WGS-assays also allow CNV detection . These NPLAG1 locus as SRS-disease-causing. In addition, the distal 1q21 region can be regarded as another SRS candidate region, and should therefore be included in the routine diagnostic workup of SRS.The two reported cases with SRS features corroborate the molecular heterogeneity of the disease; also our second case confirms the First line molecular testing for SRS comprised methylation-specific multiplex ligation probe dependent amplification  to identify the characteristic molecular alterations on chromosomes 11p15, 7 and 14q32. Molecular karyotyping was performed by SNP array analysis . All analyses were performed according to the manufacturers protocols.