Next–Generation Sequencing (NGS) has provided a deeper genetic understanding of acute myeloid leukemia (AML) that has been recently incorporated into AML classification and risk–stratification guidelines. Single molecular analysis has become inefficient and molecular testing based on NGS is emerging as an irreplaceable diagnostic tool in clinical settings. The PETHEMA cooperative group has constituted a nationwide NGS network with centralized analysis in seven high–skilled laboratories. The study of molecular profiles in the “real–life” PETHEMA cohort supports the increasing role of NGS on the clinical management of AML patients.ABL1, ASXL1, BRAF, CALR, CBL, CEBPA, CSF3R, DNMT3A, ETV6, EZH2, FLT3, GATA2, HRAS, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, PTPN11, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1 and WT1) showed 97% of patients having at least one mutation. The mutational profile was highly variable according to moment of disease, age and sex, and several co–occurring and exclusion relations were detected. Molecular testing based on NGS allowed accurate diagnosis and reliable prognosis stratification of 954 AML patients according to new genomic classification proposed by Tazi et al. Novel molecular subgroups, such as mutated WT1 and mutations in at least two myelodysplasia–related genes, have been associated with an adverse prognosis in our cohort. In this way, the PETHEMA cooperative group efficiently provides an extensive molecular characterization for AML diagnosis and risk stratification, ensuring technical quality and equity in access to NGS studies.Next–Generation Sequencing (NGS) implementation to perform accurate diagnosis in acute myeloid leukemia (AML) represents a major challenge for molecular laboratories in terms of specialization, standardization, costs and logistical support. In this context, the PETHEMA cooperative group has established the first nationwide diagnostic network of seven reference laboratories to provide standardized NGS studies for AML patients. Cross–validation (CV) rounds are regularly performed to ensure the quality of NGS studies and to keep updated clinically relevant genes recommended for NGS study. The molecular characterization of 2856 samples  with standardized NGS of consensus genes ( Introduction of Next–Generation Sequencing (NGS) into routine molecular diagnosis has provided deep molecular knowledge of acute myeloid leukemia (AML). These findings have allowed for the refinement of classification and risk stratification systems based on recurrent genetic abnormalities.In 2016, Papaemmanuil et al. proposed the first genomic classification of AML that identifies 11 molecular classes, each with distinct diagnostic features and clinical outcomes , new IntIn this situation, molecular analysis by single–gene techniques has become inefficient in order to provide a complete characterization of AML. In contrast, NGS represents a more sensitive tool to capture all the relevant molecular markers in one assay and is widely recommended to study the molecular landscape of this disease .NGS implementation to perform accurate diagnosis in AML is currently demanded by physicians and patients. However, introduction of NGS into clinical routine faces novel challenges . NGS reqIn order to provide comprehensive NGS studies to AML patients and to guarantee equity of access, the PETHEMA cooperative group (Programa Español de Tratamientos en Hematología) has established a nationwide network of central laboratories aimed to harmonize NGS results under consensual criteria in newly diagnosed and relapsed/refractory AML patients .This study summarized the NGS–AML project (NCT03311815), reporting quality control assays and the molecular profile of 2668 AML patients reported in the PETHEMA AML registry. We show co–occurring and mutual exclusion relationships among genes and distinct molecular profiles according to disease stage, age and sex and genomic classification in the “real–life PETHEMA cohort”.Implementing NGS studies in the routine molecular diagnosis of AML patients requires specialization, budgetary stability and logistical support. The PETHEMA cooperative group established a nationwide network of NGS studies for fast and standardized molecular diagnosis of AML. This strategy aims to provide coverage of NGS studies to 38.5 million inhabitants distributed in geographical areas ranging from 2.2 to 8.9 million habitants. For this purpose, seven centers with logistical and technical capacity for the management of a high number of samples were designed as reference laboratories for the centralization of samples submitted by PETHEMA institutions in each area. In this way, a large territory and population was covered. The platform was supported by PETHEMA in logistical management, as well as, closer and well–established relationships between the sample referral institution and the assigned central laboratory. The designated reference centers for NGS analysis concentrate a large number of AML samples, allowing for the rapid completion of the sequencing runs and their management by highly specialized staff.We show a prospective, multi–center, non–interventional and translational biomedical research, performed in seven Spanish PETHEMA reference laboratories: Hospital Universitario La Fe , Hospital Universitario de Salamanca , Hospital Universitario 12 de Octubre , Hospital Universitario Virgen del Rocío , Hospital Universitario Reina Sofía , Hospital Universitario de Gran Canaria Dr. Negrín , CIMA LAB Diagnostics .ASXL1, BCOR, CEBPA, EZH2, FLT3, IDH1, IDH2, NPM1, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, ZRSR2 and TP53). Moreover, there was a recommendation for the study of other genes with proven evidence on their relevance in AML pathogenesis .The development of the diagnostic platform required several meetings to coordinate criteria on which genes should be analyzed based on their clinical relevance in AML. After extensive bibliographic revision of current molecular basis of AML, all reference laboratories should assess by NGS the mutational status of genes that define the diagnosis and prognosis as well as guide treatment options  rounds have been performed. As previously reported, in the first and second CV rounds, minimum quality parameters [uniformity (>85%) and mean read depth of 1000X] and consensus recommendations for variant report  were stablished. For both CV rounds, 10 samples harboring 54 variants were distributed. In first CV round, VAF for all variants was >5% while in the second CV round, 5 from 30 total variants had low VAF ranging from 1.8–4.9%. In the third CV round, to explore the accuracy and reliability of low VAF variants (<5%) detection, 4 samples harboring 32 variants (11 VAF < 5%) were analyzed, according to previously established criteria  and “WHO2016 set 2” ; and also novel categories: “TP53 and complex karyotype (CK)”, “sAML1” , “sAML2” (More than one mutations in sAML1 genes including DNMT3A and TET2), “WT1”, “Trisomies”, “DNMT3A + IDH1/2”, “Not class defining mutations (mNOS)” and “No events” category. Since our study excludes acute promyelocytic leukemia, category “t” is not applicable.This new classification categorizes AML in 16 molecular classes with different prognostic values and encompass the established WHO entities: “WHO2016 set 1”  represented the 34% of PETHEMA–cohort.Based on the availability of clinical, cytogenetic and mutational data, 954 patients were eligible to assess the updated genomic classification by Tazi et al. . The mosCEBPA bZIP” (Median OS not reached at 32 months) and “NPM1” [29.0 months (95%CI 19.9–38.0)] had the best outcomes while inv(3) had the worst prognostic value [4.9 months (95%CI 0.8–9.1)]. Among new molecular classes, those patients without driver mutations “No events” (Median OS not reached at 33 months) or “Not class defining mutations” [23.3 months (95%CI 11.0–35.6)] showed the best prognostic value while “TP53/CK” [5.3 months (95%CI 2.9–7.6)], “sAML2” [12.1 months (95%CI 9.9–14.2)] and WT1 [4.0 months (95%CI 0.0–18.4)] classes had the worst OS (p < 0.001) ; multi–hit: 4.1 months (95%CI 2.9–5.3) (p = 0.088)] ” (Median OS not reached at 42 months), “< 0.001) . When we 0.088)] .p < 0.001)] and “TP53/CK” [3.5 (95%CI 2.6–4.6) (p < 0.001)] showed the highest risk of death compared to “NPM1” class. Remarkably, the new established “sAML2” [2.1 (95%CI 1.6–2.7) (p < 0.001)] and “WT1” [2.3 (95%CI 1.1–5) (p < 0.05)] categories were the next with higher risk of death ” [3.9 (95%CI 2.1–7.2) (of death .p < 0.001).We also assessed the integrated risk score based on cytogenetic and gene mutations proposed in the new genomic classification . In the p < 0.001) (p < 0.01) and 2.7  increased risk of death relative to favorable risk group  in the favorable risk group, 18.5 months (95%CI 14.3–22.7) in the intermediate group and 9.4 months (95%CI 7.8–11.00) in the adverse risk group (< 0.001) A. Intermsk group .p < 0.001) (p < 0.001)  months and 20.8 (95%CI 13.2–28.3), respectively (< 0.001) B. In con< 0.001) C.p < 0.001) and 3.5  higher risk of death relative to those patients classified in the favorable risk group (p < 0.001) compared to those classified as favorable risk. No statistically significant results were found in the risk of death of intermediate risk patients: 1.0  (p < 0.001), intermediate: 1.8  and adverse: 2.6 .Regarding risk score, young patients classified in the intermediate and adverse risk group showed 2.6  . In genep = 0.839), intermediate  or adverse–risk patients  according to both classifications  for favorable . Noteworthy, the detection of low VAF (<5%) variants (concordance 41.2%) was also consistent with previous studies, which report that accurate detection of low VAF variants by NGS could be compromised. CV rounds results reflected the improvement of the diagnostic platform performance as the error rate decreased in variants VAF > 5% as a result of the experience gained in NGS studies. Although the second CV round included 5 variants with a mean VAF of 3.3%, in the third CV round we aim to explore the performance of NGS studies in very low VAF variants. For this purpose, we included 11 variants with a mean VAF of 1.2%. Data analysis revealed an increase of the ER between both CV rounds due to the very low VAF variants of the third CV round. Consequently, we established VAF 5% cut–off for variant reporting, although according to other specific studies, variants in hotspot regions were reported up to 1% VAF even at borderline technical quality ,17,18.Our results reflect that a comprehensive NGS approach is suitable for defining the molecular profile of AML as over 96% of patients of the “real–life” PETHEMA cohort harbored at least one mutated gene. Our study also reflects the genomic heterogeneity that encompasses AML  as several gene–to–gene interactions but also co–occurrence and mutual exclusivity patterns across functional categories were described. Recently, published studies have highlighted the impact of co–mutations in modulating treatment response ,20.FLT3 mutations, which showed a stability rate of 43.3% in our cohort [Similarly, distinct mutational profiles were detected among disease stages, reflecting the clonal evolution of AML ,21. In fr cohort .FLT3 mutations [In addition to mutational changes according to disease stages, specific mutational profiles have been associated to patient’s clinical characteristics, such as sex and age, with significant impact on prognosis, therapeutic allocation and disease monitoring . In factutations ,31,32,33utations ,35,36. Iutations ,38.p = 0.018). In this sense, further studies are needed in order to certainly clarify the impact of sAML mutations on prognosis.Molecular diagnosis of AML has shifted towards a comprehensive mutational study driven by NGS, yielding large amounts of data. Updated diagnostic (WHO and ICC) and prognostic (ELN) classifications ,4,5 inclTP53 mutations, in the PETHEMA cohort, 47 patients were included in the mono–allelic group (32.9%), while 96 were included in the multi–hit group (67.1%). Similar results were described in the Tazi et al. cohort: mono–allelic (31.1%) vs. multi–hit: (68.9%). We did not find a distinct outcome between these groups, although the sample size was limited to draw solid conclusions. Our results are concordant with Tazi et al., who concluded that the allelic state of TP53  provide no further prognostic information in AML.Regarding CEBPA should be considered as a favorable prognosis marker [CEBPA bZIP mutations is similar to the CEBPAbi subgroup reported by Tazi et al., (1.2% vs. 1.8%) and in both cases, OS analysis includes them within a favorable risk subgroup. However, CEBPAbi and CEBPA bZIP may be overlapping categories in some patients as several studies suggest that CEBPA double mutated frequently includes mutations in the C–terminal region which allocated bZIP domain [CEBPA as a prognostic biomarker may allow for more homogeneous analysis by reducing the variability in the interpretation of the biallelic character of these mutations [On the other hand, recent studies reported that instead of biallelic mutations, only in–frame mutations in the bZIP domain of s marker . In our P domain . Moreoveutations .This report reflects the efforts of the PETHEMA cooperative scientific group to adopt a nationwide strategy network of reliable and consistent NGS analyses. The establishment of consensus subset genes and the periodic CV rounds have strengthened the diagnostic network by unifying analysis criteria and decreasing reporting variability. Molecular analyses through NGS are routinely performed for AML patients and a comprehensive molecular profile of the disease is offered to clinicians in order to individualize the therapeutic strategy. Moreover, NGS results have provided a large amount of molecular data that has revealed the molecular complexity of the disease. In this cohort, mutual exclusion and mutational co–occurrences among genes and functional categories have been deciphered and a distinct molecular profile between age groups at diagnosis and sex has been detected. Moreover, clinical validation of the current genomic classification in the “real–life” PETHEMA cohort has demonstrated the correlation of the molecular subgroups with clinical prognosis, reflecting the utility of the cooperative NGS studies in routine molecular diagnostics.


Objectives: The aim of this study was to estimate the prevalence of anti-microbial resistance (AMR) carriage and its risk factors in hospitalized migrants. Additionally, the prevalence of infectious diseases was evaluated, as well as symptoms of psychological trauma.Methods: We conducted a retrospective monocentric cross-sectional study including all migrant patients recently arrived and hospitalised over a one-year period.Results: Among 101 patients, seventy-nine percent originated from Sub-Saharan Africa. The overall AMR carriage rate was 20.7% . We isolated 5/92 methicillin-resistant Staphylococcus aureus strains (5.4%) and 15/92 extended-spectrum beta-lactamase-producing Enterobacteriaceae (16.4%). AMR carriage was associated with older age, region of origin and length of migration. Rates of HIV, HBV, and HCV infection were 39.6%, 32.7%, and 5%, reflecting sampling bias linked to reasons for hospitalization. Eleven percent had serological evidence of treponemasis and 7.8% had Chlamydia trachomatis infection. Symptoms of depression or post-traumatic stress disorder were observed for more than half the patients.Conclusion: It appears essential to offer a systematic and comprehensive post-arrival screening of AMR carriage, infectious diseases and psychological trauma to subjects who experienced migration. Emergence and dissemination of multi-drug resistant (MDR) and extensive drug-resistant (XDR) bacteria are an increasing public health problem, notably for extended-spectrum beta-lactamase producing Enterobacteriaceae (ESBL-E) . InternaMigrants are exposed to an increased risk of carriage of MDR bacteria and various other infectious diseases due to supposed higher prevalence rates in countries of origin , adverseStaphylococcus aureus (MRSA)) carriage range between 20% . To our knowledge, this is the first such study to be carried out in France and reporting the prevalence of AMR in migrants. AMR carriage in the general French population has been estimated around 6% in 2011 . Recentl.4; 28.9%E-ESBL were the most frequent MDR bacteria isolated (16.3%), as previously reported . To our p = 0.033) and a trend towards a higher risk in cases of shorter duration of migration (p = 0.074). This suggests that the country of origin may be an important marker for AMR carriage in newly-arrived migrants. The importance of the country of origin was previously described in a study in Switzerland, where a higher risk of ESBL-E carriage for patients was reported in patients originating from the Middle East  and 60.4% [95% CI: 50.4–70.5%] of patients, respectively. This is higher than was previously reported in the literature: from 5 to 30% for depression and from 10 to 30% for PTSD . PsycholThe main limitations of our study are the small sample size and the retrospective nature that implies some missing data. Due to the low sample size of our study and the number of missing data for some co-variables, our study may be underpowered and therefore, have underestimated the prevalence of studied conditions and the association between ESBL carriage and some exposure variables. All the screening tests are routinely performed and correspond to the usual practice of our department, which minimizes the risk for recruitment bias and limits the amount of missing data. It is important to notice that the patients included in our study were specifically referred to our department for medical care, and therefore are not representative of the general migrant population. However, reasons for hospitalization were not directly related to the studied conditions which minimizes the risk for recruitment bias. Furthermore, as recommended in France , AMR carriage research was performed as soon as the patients are hospitalized in order to reduce any over risk directly related to hospital settings. Screening patients in first referral medical centers would improve the sensitivity of these analyses.The European Center for Disease Prevention and Control recently published guidelines on screening for infectious diseases in newly-arrived migrants . This stFinally, more comprehensive data regarding the global prevalence of AMR carriage are required, particularly in low-income countries where increased use of antibiotics is likely to increase the prevalence the prevalence of MDR bacteria. This would allow for a better understanding of the dynamics of dissemination of AMR carriage on a global scale and mitigate the risk of the spreading of an AMR pandemic.With regards to the high prevalence of AMR, infectious diseases and psychological disorders found in this population of migrants, it appears essential to offer a systematic and comprehensive post-arrival screening of infectious diseases and signs of psychological trauma to all subjects who experienced migration.


What is the order of magnitude for evolutionary processes to be observed and which are the tools that enable us to study the viable paths in mutational evolutionary landscape?Evolution is a phenomenon traditionally studied at different scales of magnitude. In the last century, the community put a great emphasis in connecting the results obtained at different scales to build our current understanding of the subject. The building blocks of this knowledge are experiments executed at the smallest molecular scale to prove fundamental concepts. These concepts are then connected and interweaved with previous knowledge to perfect the current models of evolution.In this Research Topic we explored the process of generating molecular evolution where artificial mutagenic pressure is used to create a diverse library of mutants and the tools used to generate and analyze such diversity.Arthur M. Lesk gives his perspective on the methods to determine the three-dimensional structure using correlated mutations emerged during evolution (In this Research Topic, volution . The artMonti et al. use Zyggregator (S. cerevisiae is less toxic if compartmentalized in an aggregate, while an endogenous proteins, such as TEM-1 beta lactamase in E. coli, is functional when aggregation is avoided.In a related article, gregator , an algogregator  for whicAlejaldre et al. focus on a different aspect of protein evolution: catalytic speed and substrate specificity. Despite showing vastly differing patterns of protein dynamics at the timescale of catalytic turnover (Similarly using the TEM ß-lactamase model, turnover , all theNarayanan and Procko analyze and compare the way deep mutational scanning or deep mutagenesis have been influencing the study of the interaction between viral glycoprotein and their host receptors (Finally, in a review influenced by the recent SARS-CoV-2 pandemic and previous epidemic emergencies, eceptors . The autTaken together these articles provide a snapshot of the current Research on evolution at the molecular level, showing the relationship between evolution and physico-chemical properties of proteins and their dynamics. Most importantly, these works generate information that link protein folding and interactions.We hope that the articles collected in this Research Topic are able to convey the multifaceted nature of evolution at the molecular level and would be valuable to future scholars to understand the multiple layers of information that can be generated by mutagenesis.


There are at least four forms of Kaposi’s sarcoma (KS) with the ‘HIV’-related form being the most aggressive and can involve mucosae or visceral organs. Kaposi’s sarcoma-associated herpes virus (KSHV) is the underlying cause of this disease. It can infect endothelial and/or mesenchymal cells and establish a latent phase in host cells in which latency proteins and various non-coding RNAs (ncRNAs) play a complex role in proliferation and angiogenesis. It also undergoes periods of sporadic lytic reactivation that are key for KS progression. Complex interactions with the microenvironment with production of inflammatory cytokines and paracrine signaling is a standout feature of KS development and maintenance. KSHV impairs the immune response by various mechanisms such as the degradation of a variety of proteins involved in immune response or binding to cellular chemokines. Treatment options include classical chemotherapy, but other novel therapies are being investigated.Kaposi’s sarcoma (KS) is a heterogeneous angioproliferative tumor that generally arises in the skin. At least four forms of this disease have been described, with the ‘HIV’-related form being the most aggressive and can involve mucosae or visceral organs. Three quarters of KS cases occur in sub-Saharan Africa (SSA) as geographic variation is explained by the disparate prevalence of KS-associated herpes virus (KSHV), which is the underlying cause of this disease. It can infect endothelial and/or mesenchymal cells that consequently transdifferentiate to an intermediate state. KSHV establishes a latent phase in host cells in which latency proteins and various non-coding RNAs (ncRNAs) play a complex role in proliferation and angiogenesis. It also undergoes periods of sporadic lytic reactivation triggered by various biological signals in which lytic stage proteins modulate host cell signaling pathways and are key in KS progression. Complex interactions with the microenvironment with production of inflammatory cytokines with paracrine signaling is a standout feature of KS development and maintenance. KSHV impairs the immune response by various mechanisms such as the degradation of a variety of proteins involved in immune response or binding to cellular chemokines. Treatment options include classical chemotherapy, but other novel therapies are being investigated. In 1872, Moritz Kaposi, a Hungarian physician, first noted multifocal pigmented skin lesions in elderly European and Mediterranean men. He was describing what is now known as the “classic” form of Kaposi’s sarcoma (KS) . IncidenThe “epidemic” HIV-related form is particularly aggressive and can involve mucosae or visceral organs ,9. Compa6/year. In a recent epidemiological nationwide study conducted in France, the incidence of KS was 2.5/106/year, with 18% occurring in an HIV context, and 28% in patients with immunodepression [6/year, higher in the Italian region (Veneto) as compared to two other French regions  [The incidence of KS varies considerably around the world ,14. Accouitaine) .This geographic variation of KS is driven by the disparate prevalence of KSHV, which is the underlying cause of all forms of KS, and HIV infection that worsens the carcinogenic outcome of KSHV infection . The preKSHV, also known HHV8, is a double-stranded linear DNA gamma-herpesvirus and is the causal agent of all forms of Kaposi sarcomas. It is also the underlying cause of primary effusion lymphoma (PEL) and multicentric Castleman Disease (MCD) . A link Similar to other herpesviruses, KSHV establishes latency in host cells , responsible for lifelong infection. During latency, only a few genes from the latency locus are expressed, such as open reading frame (ORF)71 (v-FLIP), ORF72 (v-Cyclin), ORF73 , and the K12 region  . LANA biKSHV affects endothelial cells and/or mesenchymal cells that consequently adopt an endothelial differentiation. Early lesions (plaque or patch) often appear as a granulation type reaction with immune cell infiltration, intense angiogenesis, and proliferating “spindle”-shaped cells of endothelial and macrophagic cell origin, which are the tumor cells of KS. A typical KS is characterized by proliferation of spindle-shaped endothelial cells in the skin and sometimes in mucosal and visceral sites. Kaposi cells form slit-like vascular spaces, with often extravasated inflammatory cells and red blood cells. In late, nodular, lesions of KS, tumor cells eventually become the predominant cell type and lesions acquire a fibrosarcoma-like aspect, though neoangiogenesis is present . KS cellSpontaneous reactivation of KSHV in lymphatic endothelial cells is important to maintain and expand the population of infected cells in the tumor and to promote tumor cell expansion. The mechanisms of activation of the lytic stage in these cells may involve specific transcription factors. Prox1 is a lymphatic endothelial cell-specific transcription factor that induces the expression of the RTA gene to promote lytic cycles, while SOX18, another transcription factor necessary for lymphatic endothelial cell development, promotes an increase in the number of latent genomes. Both are induced by KSHV infection and play an important role in KS oncogenesis, Prox1 being necessary to maintain the number of infected cells in the tumor ,36.Oncogenic DNA viruses produce oncogenic proteins responsible for cellular proliferation and the protection of the host cell against apoptosis. They evade the host immune surveillance system by multiple mechanisms such as the reduction of viral gene expression to limit the amount of viral antigen presentation, the synthesis of critical proteins affecting the process of antigen presentation, and the expression of various viral “immunoevasins” that manipulate host cells to reduce immunogenicity ,38 Tabl.Following KSHV infection of endothelial cells , it establishes a latent phase, expressing a limited set of genes, including LANA (ORF73), vFLIP (ORF71), vCyclin (ORF72), Kaposins (ORFK12), and a set of miRNAs. Some of these proteins are analogs of cellular genes  and have oncogenic properties in vitro in cell cultures ,38.These latent proteins, along with the miRNAs, play a complex role in cell survival, proliferation, and angiogenesis. They promote the tumorigenesis of KS. ORF72 (vCyclin) expression overcomes senescence in infected endothelial cells and activates a telomere maintenance mechanism essential for the oncogenic process in KSHV-infected cells ,40. LANALytic cycles of KSHV are observed in lymphatic and endothelial cells and in KS cells. They play a key role in the progression of KS in the clinic . The indSeveral lytic stage proteins modulate host cell signaling pathways to allow cell survival and proliferation and to augment viral replication . In the The oncogenic proteins of KSHV thus activate a series of cellular pathways, the MAPK, NFkB, and PI3K pathways, as well as the production of proinflammatory cytokines and angiogenic factors, which play a key role in the oncogenesis of KS. One of the intriguing features of KSHV-induced oncogenesis is the contribution of soluble factors.The infection of endothelial cells with KSHV results in cell transformation, accompanied by complex interactions with the microenvironment . InfecteThe previously mentioned lytic gene ORF74, encoding for the constitutively active vGPCR, activates MAP kinase and NFkB pathways , inducinAll these elements are responsible for the production of inflammatory cytokines, with paracrine and endocrine paraneoplastic activities.The immune system plays an important role in the control of KS . RestoraKSHV produces proteins and miRNAs that impair cytotoxic T or NK cell-mediated killing . KSHV K3The inhibition of immune response induced by KSHV occurs at multiple steps of the development of the immune response, from the initial activation of innate immunity. KSHV viral interferon regulatory factors (vIRFs) impair TLR signaling at multiple levels: vIRF reduce TLR3-mediated interferon induction . The RTAKSHV induces the production of viral cytokines and chemokines while inducing the production of cellular inflammatory cytokines. This is observed in vitro when monocytes are infected by KSHV, and in vivo in patients with KS. These increased serum levels of IL-6, IL-10, and TNF, and detectable vIL-6, are indeed observed in patients with progressive KS . These cKSHV also contains three viral homologs of cellular chemokines, vCCL1 to vCCL3 . vCCL2 binds to a wide variety of chemokine receptors on many different cell types and can have agonistic or inhibitory effects. vCCL1 binds to CCR8, which is prominently expressed on Treg cells, suggesting that the virus may recruit this T cell subtype . vCCL2 hAs mentioned previously, there is a high individual variability in disease development and presentation within all forms of KS. Genetic differences between populations could explain this heterogeneity, suggested by the observation of familial clustering of KS cases . PolymorTo what extent can the partial understanding of the oncogenesis of KS guide us to new treatment decisions?There are multiple active treatments of KS. While classic chemotherapy has been used for decades, many clinical trials evaluating immune checkpoint inhibitors or anti-angiogenic agents are underway in both HIV and non-HIV-related KS. The treatment of KS in the clinic is based on consideration of multiple criteria including patient’s comorbidities and preferences, disease extension, and the immunological and virological status of the patient . Since KIf insufficient, e.g., in advanced or very symptomatic disease, chemotherapy can be considered. Pegylated liposomal doxorubicin (PLD) is a standard first line . PaclitaOther front-line options include docetaxel and nab-paclitaxel as small studies have shown comparable efficacy and acceptable toxicity ,87. OralWith the identification of the role of endogenous cytokines, angiogenic factors, and immune suppressive factors in KS progression, anti-angiogenic agents & immunomodulators are investigated ,97,98,99Pomalidomide is an immunomodulatory agent capable of reversing viral-induced downregulation of immune surface receptors, rendering KS cells more immunogenic. Promising results were observed in prospective trials with RRs up to 40% for HIV-related KS ,93. ChroKICS is a severe life-threatening condition related to an overwhelming production of proinflammatory cytokines . TreatmeTargeting viral miRNAs has promising potential as viral miRNAs are distinct from cellular miRNAs. In a recent study, targeting three KSHV miRNAs with antisense inhibitors suppressed the growth of KSHV lymphoma cells in mouse xenograft models .The understanding of the mechanisms of cellular transformation due to KSHV in KS has considerably progressed 28 years after the identification of the virus. Multiple steps and viral oncoproteins are involved in the transformation process, which requires a complex interplay between the viral oncoproteins and cellular proteins as well as the modulation of the host’s immune system response. This opens new avenues for the treatment of this multifaceted disease that affects large human communities across the globe.


Integrating radical (open‐shell) species into non‐cryogenic nanodevices is key to unlocking the potential of molecular electronics. While many efforts have been devoted to this issue, in the absence of a chemical/electrochemical potential the open‐shell character is generally lost in contact with the metallic electrodes. Herein, single‐molecule devices incorporating a 6‐oxo‐verdazyl persistent radical have been fabricated using break‐junction techniques. The open‐shell character is retained at room temperature, and electrochemical gating permits in situ reduction to a closed‐shell anionic state in a single‐molecule transistor configuration. Furthermore, electronically driven rectification arises from bias‐dependent alignment of the open‐shell resonances. The integration of radical character, transistor‐like switching, and rectification in a single molecular component paves the way to further studies of the electronic, magnetic, and thermoelectric properties of open‐shell species. A verdazyl radical, stable in its open‐shell state at ambient conditions, has been used to fabricate single‐molecule devices that show enhanced charge‐transport efficiency, electrochemical transistor behaviour, and rectification. All the properties arise from the presence of an unpaired electron, strongly localised on the verdazyl core. T(E) (the probability for an electron with given energy E to tunnel through the molecular wire) near to the electrode Fermi level.G of a molecular junction can be estimated using the Landauer formula G = G0T(FE) where G0 = 2e2/h ≅ 77.48 μS, a high transmission coefficient (i.e. a resonance) at the Fermi level results in high charge transport efficiency. The high slope of T(E) vs E near the Fermi energy offered by radical systems is also predicted to give rise to enhanced Seebeck coefficients,+)Molecular radicals feature open‐shell electronic structures (i.e. an odd number of electrons) and, as a consequence, they are often highly reactive and sensitive to atmospheric oxygen and moisture. Nevertheless, since Gomberg's successful synthesis of the triphenylmethyl (trityl) radical in 1900,1 maintains its radical character when fabricated into single‐molecule junctions between two Au electrodes, showing significantly enhanced conductance when compared with its closed‐shell tetrazin‐3‐one precursor 2  and 27 600 cm−1 , assigned to transitions involving singly‐occupied orbitals,[2 only shows an absorption band in the ultraviolet corresponding to a π–π* (HOMO–LUMO) transition , consistent with the calculated distribution of the spin density over these atoms. EPR measurements of 1 adsorbed on a Au substrate confirm the retention of its open‐shell character  method in order to characterise their charge‐transport properties.V until a metallic contact having conductance ≫G0 is formed. The tip is then withdrawn at a constant speed (10 nm s−1 in this study) in a dilute solution of the target molecular wire, while continuously monitoring the current I at high acquisition rate (>10 kS s−1) as a function of the relative tip‐substrate position z. During the withdrawal process, the metallic contact is continuously thinned until it is reduced to a point contact having conductance G0. As the withdrawal continues, the point contact will break, generating a nanogap of ≈0.5–0.6 nm due to snapback of the electrodes.G(z) traces  that bear multiple plateaux at: (i) integer multiples of G0, characteristic of conductance quantisation of single‐channel conductors such as Au atoms; and (ii) at values ≪G0, characteristics of transport through partially open quantum channels such as molecular wires. Data are then compiled in semilogarithmic histograms and heatmaps for further statistical analysis. Plateaux in the G(z) traces result in peaks in the histograms and in high‐density areas in the heatmaps, yielding information on the most probable conductance values and molecular extension of the junction. Information about the instruments and materialsSingle‐molecule junctions of 1 displayed efficient charge transport, with a conductance peak centred at 10−3.4 G0, almost one order of magnitude more conductive than its closed‐shell precursor 2 (10−4.3 G0) . As compounds 1 and 2 have similar molecular geometry and share the same linkers to the electrodes (thioanisole), no significant difference in the structure of the junction is expected and differences in molecular conductance are therefore attributed to differences in the electronic structures of the heterocyclic molecular cores.The 6‐oxoverdazyl radical E through junctions formed from 1 and 2, details of which are given in the Supporting Information. A pair of sharp resonances in the transmission function T(E), lying close to the Fermi level, are calculated for the junction formed from 1. These resonances arise from the singly‐occupied  and singly‐unoccupied  orbitals of the radical, as also demonstrated by local density of state calculations (see Supporting Information). Our calculations of 1 between gold electrodes  for 1 above 10−3 G0 over a wide range of electron energies around the Fermi level. In contrast, the transport profile of the closed‐shell tetrazin‐3‐one 2 shows that the Fermi level lies towards the bottom of a dip in the transmission function, within a wide HOMO–LUMO gap  and one‐electron reduction (to the monoanion) processes  and a Pt counter electrode vs an Ag/AgCl reference electrode. The STM tip was insulated with Apiezon wax+) redox couple. The conductance of 1 remained at values around those obtained without electrochemical control from −0.2 to −0.8 V vs Fc/Fc+. In this potential window, a small electrostatic gating effect can be observed, with conductance slowly increasing as the potential is made more negative .To verify the involvement of the well‐aligned singly‐occupied orbital in the increased charge transport efficiency of e Figure c. As the1 offers a resonance lying close to the Fermi level in the junction, with the abrupt decrease in junction conductance at more negative gate potentials indicating that this resonance is removed when 1 is reduced. In the potential window where 1 is in its open‐shell state (−0.2→−0.8 V vs Fc/Fc+), conductance increases as the potential is made more negative, indicating that the transport resonance due to the SUMO (or β‐LUMO) is initially at energy higher than the Fermi level of the electrodes. As the gate potential passes −1.0 V vs Fc/Fc+ and 1 is reduced, the slope of the conductance/potential dependence suddenly changes sign, and the transport resonance is now due to the HOMO of the reduced 1, at energy significantly lower than the Fermi level. Conductance data for 1 in the full electrochemical window explored (−1.3→0.5 V vs Fc/Fc+) is provided in the SI. Attempts to obtain single‐molecule conductance data at potentials >0.5 V vs Fc/Fc+ (where 1 would be oxidised to the monocationic state) were not possible due to increased instrumental noise at these potentials. We hypothesise this is due to the insolubility of the cationic species in the ionic liquid used in this study, which therefore precipitates from solution preventing further junction fabrication. Changes in the properties of ionic liquids in high electric fields[1 in 1‐butyl‐3‐methylimidazolium triflate (available in the Supporting Information). In this environment, the reduction of 1 to the anionic state shows a quasi‐reversible behaviour, but its oxidation is completely irreversible, with only the forward wave +1→1 appearing during cyclic voltammetry.The initially observed electrostatic gating effect provides confirmation that ic fields40, 411 is enhanced by the presence of a partially filled molecular orbital near the Fermi level, attention was turned to single‐molecule bias modulation experiments, with the aim of establishing the current‐voltage (I–V) behaviour of this compound. In these experiments, a modified STM‐BJ technique was used. The tip was driven into the substrate and then retracted in a staircase manner, with steps of size commensurate to the length of the molecule of interest . Between steps, the tip‐substrate bias was held at a stable value (+200 mV) for 25 ms, then stepped and ramped between +2 and −2 V at a rate of 80 V s−1 to obtain each I–V curve over a period of 50 ms; at the end of the ramp, the bias was stepped back to 200 mV and again held stable for further 25 ms. Data were then sliced and processed using automated algorithms described in the Supporting Information. The I–V characteristics of 1 are not linear, with the ohmic behaviour displayed between ±0.7 V giving way to clear evidence of current rectification above ±1.5 V . It is worth noting that the I–V experiments performed here lack the coated tips and highly polar environment required to drive 2‐terminal (2‐electrode) electrochemistry[1 does not change during these measurements.Having established that charge transport through V Figure a, b. At 1 enters the bias window at positive bias voltages occupied) orbitals of 1 or the frontier orbitals of 2, as the charge density distribution is different for different orbitals. For example, the charge density of the α‐SOMO is localised mainly on the verdazyl ring whereas that of the β‐SOMO is localised mainly on the aurophilic termini (Table S2 in the Supporting Information) and therefore the shift of resonances due to the applied bias for these orbitals does not follow the same trend. These phenomena combined lead to a distinct asymmetry of the I–V behaviour for the open‐shell 1. Furthermore, the rectifying behaviour in our study is demonstrated by a structurally symmetrical molecule therefore arises within the framework of a coherent tunnelling mechanism, and is not due to the surrounding environment[2, where the Fermi energy is close to the middle of the HOMO–LUMO gap as is often the case in molecular junctions, the shift of transmission spectra by bias voltage does not lead to significant difference of the transmission coefficient near FE at biases of opposite polarity, and therefore there is no asymmetry in the I–V behaviour  and semi‐unoccupied (SUMO or β‐LUMO) molecular orbitals. The energy alignment of these resonances with the Fermi levels of the metal electrodes grants 1 significantly enhanced charge‐transport efficiency compared to its precursor 2. The radical compound 1 can be reversibly electrochemically reduced to a closed‐shell anion within the junction, with the distinct charge‐transport properties of the two charge states giving rise to single‐molecule transistor behaviour. In addition, the bias‐dependent behaviour of the SUMO transport resonance imparts rectifying behaviour to junctions fabricated with 1, with significantly greater current flowing under positive bias due to the migration of the transport resonances within the bias window. These results provide a strategy to incorporate radical compounds in single‐molecule devices, key to enabling technologies such as molecular spintronics and molecular thermoelectrics.These studies have demonstrated that the 6‐oxoverdazyl radical Supporting Information: The Supporting Information document contains details about the methods employed and additional single‐molecule conductance data and calculations.PJL, SS and HS conceived the project. VJK and JZW synthesised the compounds used in this study and performed their characterisation. AV and RJN designed the charge transport experiments. SN performed the single‐molecule experiments and analysed the data with software written by AV. SS performed the computational studies. EG performed preliminary single‐molecule measurements. XQ trained SN in the electrochemical single‐molecule experiments and assisted data acquisition. MJ and NC performed cw‐EPR measurements. All authors contributed to the discussion of the results. AV, PJL and HS wrote the paper with contributions from all authors.Raw single‐molecule charge transport data, Labview Vis, and Python code used for its processing are available under a CC‐BY license in the University of Liverpool Data Catalogue at:STMBJ and EC‐STMBJ measurements)DOI: 10.17638/datacat.liverpool.ac.uk/1529 (I–V measurements)DOI: 10.17638/datacat.liverpool.ac.uk/1513  should be addressed to the authors.Supporting InformationClick here for additional data file.


The use of agricultural waste biomass for nanocellulose production has gained interest due to its environmental and economic benefits compared to conventional bleached pulp feedstock. However, there is still a need to establish robust process technologies that can accommodate the variability of waste feedstocks and to understand the effects of feedstock characteristics on the final nanofiber properties. Here, lignocellulosic nanofibers with unique properties are produced from various waste biomass based on a simple and low-cost process using mild operating conditions. The process robustness is demonstrated by diversifying the feedstock, ranging from food crop waste (corn stover) to invasive grass species (reed canary grass) and industrial lignocellulosic residues . This comprehensive study provides a thorough examination of the influence of the feedstocks’ physico-chemical characteristics on the conversion treatment, including process yield, degree of delignification, effectiveness of nanofibrillation, fiber morphology, surface charge, and density. Results show that nanofibers have been successfully produced from all feedstocks, with minor to no adjustments to process conditions. This work provides a framework for future studies to engineer nanocellulose with specific properties by taking advantage of biomass feedstocks’ intrinsic characteristics to enable versatile applications. Nanocellulose products are primarily produced from bleached wood pulp, a high-purity, expensive feedstock comprising virtually pure cellulose . CurrentThere is increasing interest in using low-purity biomass waste feedstocks such as agricultural residues and forest residues, which contain other components besides cellulose , to produce nanofibers due to their lower cost compared to bleached pulp ,7. UsingThe intrinsic characteristics of each of the three biopolymers will influence the properties of the final product without requiring additional, and often expensive, post-processing modification reactions. Hemicelluloses are branched, low molecular weight heteropolysaccharides whose specific chemical composition varies with different plant species. Hemicelluloses commonly confer colloidal stability, easier fibrillation, and negative charge to nanofibers ,10,11. L2 emissions than nanofibers produced using state-of-the-art methods such as TEMPO oxidation  cmNanofiber morphology was examined by scanning electron microscopy (SEM) and atomic force microscopy (AFM) techniques. AFM images of LCNFs were collected using a Bruker ICON AFM in contact mode and a scan rate of 1 Hz. LCNF suspensions were diluted to 0.001 wt.% with DI water and bath sonicated for 5 min to promote dispersion. An amount of 100 µL of LCNF was drop-casted onto a freshly cleaved mica disc previously coated with 50 µL of L-lysine, rinsed with DI water, and air-dried. The LCNF width and length were computed from at least 20 measurements of individual fibrils. SEM images of LCMFs were collected using an Apreo S (Thermofisher Scientific) coupled with a standard ETD in-chamber SE detector and a T2 in-column SE detector. LCMF suspensions were diluted to 0.01 wt.% with DI water and bath sonicated for 3 min; then, 10 µL of sample was drop-casted onto a clean SiO2 wafer. Before imaging, the samples were sputter-coated with a 4 nm thick platinum layer using a Leica EM ACE600 coater. Operating conditions were at high vacuum with an acceleration voltage of 2 kV and beam current of 6.3 pA. The LCMF width was computed from at least 20 measurements of individual fibrils. Unfortunately, it was not possible to measure LCMF fiber length due to the entanglement of fibers in the SEM imaging method.The effects of pulping and PAA pretreatment reactions on the main chemical components of the different biomass feedstock are represented in Extensive delignification during pulping was observed for both CS and RCG, with similar delignification percentages ranging from 78–81% , while IH had the lowest delignification of only 36%  B. The loAfter PAA pretreatment, all feedstocks displayed a trend of further delignification accompanied by minor carbohydrate losses, comparable to previous studies employing PAA treatments on different feedstocks ,43,44. TCompared to the untreated biomass, both CS and RCG had lower lignin recoveries (5–7%), while IH still retained 28% of the original lignin in its composition B. These −1) than those from CS and RCG (110–112 µmol g−1) (−1 in FTIR spectra of both IH-LCNF and IH 10×-LCNF compared to that of CS and RCG. The band at 1604 cm−1 has been previously associated with carboxyl groups present in hemicellulose [−1 corresponding to C=C stretching vibration of lignin aromatic rings [mol g−1) A. A simimol g−1) B. The hiellulose ,51 that ic rings  was more−1), the CD values reported in this work for CS, RCG, and IH (110–344 µmol g−1) are relatively lower due to the milder oxidation reaction that occurs during PAA pretreatment, as previously reported by our group [When compared to nanofibers produced via harsher reactions such as TEMPO-mediated oxidation  associated with the carboxyl groups of glucuronic acid in hemicellulose [−1 and around 1732 cm−1 appeared exclusively in IH pulp, representing molecules that are inherent to this specific type of biomass. The band at 780 cm−1 has been previously assigned to the carboxyl groups of hemicellulose [−1 has been attributed to C=O stretching vibration of either acetyl groups present in xyloglucan (a specific type of hemicellulose present in IH biomass) [To further elucidate the origin of the higher CD of both products made from IH biomass, FTIR spectra of the pulps after alkaline peroxide pulping  were also collected . This adellulose ,51. In aellulose , while tbiomass) ,56 or cabiomass) ,58.The above results show that IH’s higher carboxyl content, compared to CS and RCG, is mainly associated with hemicellulose acid groups and the presence of pectins. The published literature observed that IH biomass has a high galacturonic acid content attributed to the presence of pectin molecules such as rhamnogalacturonan-I ,60. Corr−1 has been assigned to C-O stretching of C5 substituted aromatic rings, such as syringyl and condensed guaiacyl units of lignin [Finally, the FTIR peak at 1317 cmf lignin ,51. AlthThe lower LCNF yields of samples prepared from IH compared to CS and RCG demonstrate a lower degree of nanofibrils released from the bigger fibrils in the original hierarchical structure of IH. This outcome results from the physical structure of IH biomass, which comprises bast and core fibers. Bast fibers are incredibly long, about 25 mm in length , while core fibers have similar physical characteristics as hardwoods, being 0.8 mm in length . The divThe crystallinity index (CI) of the different lignocellulosic nanofibers is also included in The morphology of the LCNF fractions generated from different biomass feedstocks was examined by AFM imaging , and theThe morphology of the LCMF fractions was characterized by SEM imaging , and theThe present study demonstrates that lignocellulosic nanofibers may be successfully produced from corn stover, reed canary grass, and industrial hemp via the same conversion process using mild conditions. The process was proven robust, generating products with similar morphologies despite widely different feedstocks and offering a practical pathway to manufacture lignocellulosic nanofibers from other agricultural waste biomass such as wheat straw, rice straw, rice husk, sugarcane bagasse, and switchgrass for example. This work also reported how the feedstocks’ physico-chemical characteristics influenced the final nanofibers’ properties. A feedstock with physical characteristics similar to woody materials (IH in this study) was more difficult to delignify under the mild reaction conditions, resulting in nanofibers with higher lignin recovery (28% recovery) compared to other feedstocks (5–7%); but increasing the chemical loading during PAA pretreatment resulted in higher delignification of IH (6% lignin recovery) with minor carbohydrate loss. IH’s unique physical structure (comprising bast and core fibers of vastly different sizes) also affected the efficacy of the mechanical treatment step, impacting the nanofibers’ separation yields and morphology. Finally, feedstocks with large amounts of glucuronic acids in hemicellulose and/or pectins produced nanofibers with greater anionic surface charge (up to three times higher charge density than from other feedstocks).Ultimately, using waste biomass feedstocks instead of bleached pulp enables engineering the nanofiber properties due to the presence of cellulose, hemicellulose, and lignin, where each can provide distinctive properties to the final product. We use nature’s inherent characteristics to generate nanofibers with specific properties instead of expensive post-processing surface modification reactions. The present process also allows for customization of the nanofiber properties by tuning the reaction condition parameters. In the long run, using low-cost waste feedstocks available in different regions of the United States can provide substantial economic and sustainability benefits to nanocellulose production, presenting a significant stride toward large-scale production and commercialization for various applications.


In this work, we report the obtaining of new hybrid nanocomposites with catalytic activity formed by nanofibers of polymer blends and gold nanoparticles. The nanofibers were obtained by electrospinning blends of a poly (ionic liquid) (PIL) and its precursor polymer, poly (4-vinyl pyridine) (P4VPy). The characteristics of the nanofibers obtained proved to be dependent on the proportion of polymer in the blends. The nanofibers obtained were used to synthesize, in situ, gold nanoparticles on their surface by two-step procedure. Firstly, the adsorption of precursor ions on the nanofibers and then their reduction with sodium borohydride to generate gold nanoparticles. The results indicated a significant improvement in the performance of PIL-containing nanofibers over pure P4VPy NFs during ion adsorption, reaching a 20% increase in the amount of adsorbed ions and a 6-fold increase in the respective adsorption constant. The catalytic performance of the obtained hybrid systems in the reduction reaction of 4-nitrophenol to 4-aminophenol was studied. Higher catalytic conversions were obtained using the hybrid nanofibers containing PIL and gold nanoparticles achieving a maximum conversion rate of 98%. Remarkably, the highest value of kinetic constant was obtained for the nanofibers with the highest PIL content.  The rapid development of nanoscience and nanotechnology in recent decades has led to significant advances in the applications of nanomaterials in many technological fields. Thus, the special physical and chemical properties of nanomaterials have been widely explored to develop or improve applications aimed at solving important current global challenges, such as environmental remediation, sustainable energy, and climate change. Indeed, a great deal of scientific activity has been developed to obtain nanomaterials able to contribute, for example, to capture and efficiently use carbon dioxide from the air, degrading toxic pollutants in water and contributing to process of energy conversion and storage ,2,3. ThuThanks to the important achievements of nanoscience and nanotechnology, it is now possible to prepare with great precision many catalytic materials at the nanometer scale, achieving a significant improvement in their performance thanks to the so-called “nanoeffects”. These nanoeffects, although not fully understood to date, are attributed to structural, electronic and quantum size effects . NanocatNotwithstanding the above, the use of nanomaterials as catalysts also presents some difficulties. For example, a considerable decrease in the activity of catalytic NPs has been observed after a few cycles of use, which has been attributed to aggregation and surface passivation phenomena. Indeed, when NPs agglomerate, they tend to lose their nanometer size and transform into bulk materials losing their special properties. Another problem really challenging is the difficulty to recover the catalytic nanoparticles from the reaction medium for further use. To solve these problems a suitable strategy is to support the nanoparticles on structures of different type and nature , achieviIn general, it is desirable that the ideal supports are chemically inert, have a large surface area and uniform size, and retain the nanocatalyst efficiently . Among tInspired by the above, we have prepared a hybrid nanomaterial consisting of polymeric nanofibers and gold nanoparticles, and we studied its performance as a heterogeneous catalyst using the well-known reduction reaction of 4-nitrophenol (4NP) as a model. Noticeably, in this simple reduction reaction with sodium boron hydride catalyzed by gold nanoparticles, 4-NP, a compound considered a priority pollutant by the USEPA, is effectively reduced to form the considerably less toxic compound 4-aminephenol (4-AP) under mild reaction conditions, and the reaction can be easily monitored by UV-vis spectroscopy ,15.2−) and poly 4-vinyl pyridine PV4Py, while the gold nanoparticles were synthesized in situ on the nanofibers by reduction in ionic precursors adsorbed on them. The polymeric ionic liquids PILs, also known as poly (ionic liquids) are a special type of polyelectrolytes that contain some of the chemical groups of ILs, cations and anions. PILs keep some of the special properties of Ils, such as, negligible vapor pressure, ionic conductivity, thermal stability, tuneable solution properties, nonflammability, and high chemical and electrochemical stabilities, as well as good compatibility. Many of the special properties that characterize ILs are mainly attributable to their ionic nature and are, therefore, also present in PILs. A particularly interesting property of PILs is their high capacity to interact and their compatibility with different materials, both organic and inorganic, hybrids, e.g., metal-organic and biomacromolecules 0 and [4NP]t correspond to 4-NP concentration at the beginning of the reaction and at t minutes after this was started, respectively, t is the reaction time, and appk is the pseudo first-order kinetic constant of the reaction. Using the Lambert–Beer relation, Equation (3) can be converted to Equation (4).tAbs and 0Abs correspond to 4-NP absorbance at the beginning of the reaction and at t minutes after this started, respectively. The kinetic data were determined using a pseudo-first order kinetic model, as Equation (3) indicates.appk values, both for pure P4VPy nanofibers and with different poly liquid ionic contents, containing in all cases gold NPs, are shown in The results obtained, conversion profiles, pseudo first order models, and In all cases, a rapid increase in the conversion percentage was initially observed, which slowed down as time progressed, until the highest conversion values were reached after 140 min of reaction. Interestingly, the highest value of 4 NP to 4 AP conversion (96%) was obtained with the nanofibers with the highest PIL content.app, corresponding to 10.5%, was observed for the reaction catalyzed with the nanofibers with 30% w/w of PIL and gold NPs, with respect to those with only P4VPy and gold NPs. Additionally, although the values of the kinetic constants are of the same order, an evident increase in the magnitude of k4−, through an exchange phenomenon with the counterion NTf2−. Hybrid systems, thus obtained, (NFs-Gold NPs) showed catalytic activity in the reduction reaction of 4-nitrophenol to 4-aminophenol using sodium borohydride. In terms of catalytic activity, higher conversion values were obtained with the nanofibers containing PIL and gold nanoparticles compared to those constituted of P4VPy and gold nanoparticles. Remarkably, the highest magnitude of the kinetic constant was obtained for the nanofibers with higher PIL content.PIL/PV4Py blends nanofibers with different compositions were obtained by electrospinning technique from solutions in the binary solvent DMF/DCM. Importantly, as the PIL content in the solution increased, the diameter of the NFs obtained decreased, while the amount per unit area of the same ones incremented. The process of adsorption of Au III ions and their subsequent in situ reduction, allowed to obtain Au NPs supported on PIL/P4VPy nanofibers. The presence of abundant charged groups on the polymer NFs due to the inclusion of PIL favored the adsorption process of AuClAccordingly, the inclusion and content of PILs in NFs play a fundamental role in the catalytic efficiency and performance of the Au NPs-PIL/P4VPy NFs hybrid nanomaterial. Undoubtedly, this allows envisioning potential technological applications in the field of heterogeneous catalysis for this type of materials.


Klinefelter syndrome (KS) is the most frequent sex chromosome aneuploidy in males. KS diagnosis disclosure has an important impact on diagnosis acceptance and the increase in prenatal diagnostic procedures raises questions regarding communication to children/adolescents. Limited data are currently available on this issue. The aim of the study was to investigate aspects like the best timing (when), topics (how), and healthcare professional (who), which, in the opinion of both KS patients and parents, may be considered the best for diagnosis communication to KS children/adolescents. We also analyzed how participants received the communication in real life and evaluated the differences between the responses given by parents who receive KS diagnosis before or after KS patient birth regarding disclosure of KS communication. KS adult patients, KS mothers, and KS fathers, not belonging to the same family, completed a questionnaire containing quantitative measures , open-ended questions, and multiple choice questions. Parental responses were divided according to the timing at which the communication occurred: prenatal age diagnosis (PRE-D) or postnatal age diagnosis (POST-D). A total of 41 KS adults and 77 KS parents  were recruited. Most KS patients and most POST-D parents consider that communication should be provided before 14 years of age; most PRE-D parents consider 14–18 years of age the best period for communication. We suggest that communication should occur preferably before 18 years of age by a multidisciplinary team  and that the information should deal not only fertility and hormonal aspects but also metabolic and cognitive features.The online version contains supplementary material available at 10.1007/s12687-022-00585-0. Klinefelter syndrome (KS) is the most frequent sex chromosome aneuploidy in males with an estimated prevalence of 1/660 males and is caused by the presence of one extra X chromosome to a normal karyotype   play a critical role in diagnosis communication in all fields of medicine and those dealing with KS should provide adequate counseling , discussions on diagnosis and treatment are recommended as soon as the child is able to understand , which topics (how) and the most suitable HCP to communicate the diagnosis to KS children/adolescents as well as individual opinions on emotions generated among the listeners  by sharing the diagnosis. We also analyzed how KS adult patients and KS parents received communication of the genetic diagnosis in real life , and then we evaluated the differences between the answers of parents who receive KS diagnosis before or after KS patient birth regarding disclosure of KS communication.Individuals aged 18 years old and older with blood karyotype 47, XXY, and KS mothers/fathers were recruited during endocrinological examinations and/or through the support of the “Nascere Klinefelter” Association.–demographic features–the timing of diagnosis communication–topics discussed during diagnosis communication–HCPs who communicated the diagnosis–individual opinions on when and how the diagnosis should be disclosed and who should do it–individual opinions on emotions generated among the listeners  by sharing diagnosisParticipants were invited to complete a self-administered and anonymous questionnaire based on open-ended questions, multiple-choice questions, and statements based on quantitative measures . There was no financial compensation for the participants. This study was approved by the local ethic committee. Informed consent was obtained from all patients. The questionnaire was edited on the basis of published evidence concerning the communication of diagnosis of sex aneuploidies  Dalkey . For mulP values < 0.05 were considered as indicating statistical significance.The Delphi method aims at reaching the best estimate of consensus and at providing recommendations on controversial topics Dalkey . ConsensA total of 41 KS adult patients and 77 KS parents, all Caucasians, completed the survey. In particular, 53 PRE-D (43 mothers and 10 fathers) and 24 POST-D parents (19 mothers and 5 fathers) were recruited into the study. KS patients and parents did not belong to the same families. Participants were asked to recall events in the past. PRE-D and POST-D parents received KS diagnosis information 14.7 ± 6.9 years and 18.5 ± 7.1 years, respectively, before participating in the survey. Moreover, KS patients had received diagnosis on average 15.4 ± 6.3 years before this study. Participant characteristics are summarized in Table p < 0.05).Most of the KS adult patients believed that diagnosis communication should take place before 14 years old 53.7%) or between 14 and 18 years of age (39%) (Table .7% or bep < 0.01). Moreover, the distribution of answers between KS patients and PRE-D parents’ answers is significantly different (p < 0.05).Most of the PRE-D fathers indicated that the diagnosis should be communicated when the patient is 14–18 years old (50%) or after 18 years of age 30%). All POST-D fathers would communicate diagnosis before 14 years of age and none after 18 years of age , whereas cognitive and metabolic syndrome topics were not fully addressed (ND/NA). PRE-D and POST-D mothers/fathers agreed they received adequate information about fertility, cognitive, and hormonal features (consensus reached), whereas metabolic issues were not completely addressed (ND/NA). Topics discussed during diagnosis communication are summarized in Table PRE-D mothers/fathers think that sharing the diagnosis with friends, schoolmates, and teachers generates fear (ND/NA), pity (ND/NA), and misunderstanding (ND/NA) but not empathy (agreement). POST-D mothers/fathers and KS adult patients disagree that sharing diagnosis creates fear, pity, and misunderstanding (consensus reached). Moreover, they agree that sharing the diagnosis causes empathy among the listeners (consensus reached). Opinions of participants are summarized in Table Almost half of KS adult patients 41.5%) would like the diagnosis to be communicated by an endocrinologist; a lower percentage reported that it should be communicated by a psychologist (17%) or by parents alone (12.2%). In the opinion of PRE-D parents, diagnosis should be communicated mostly by geneticists (39.6%) or by parents together with endocrinologists (18.9%). POST-D parents indicated that diagnosis should be communicated by parents together with a psychologist (29%), by parents alone (20.9%), or by parents together with endocrinologists (20.9%) , topics (how), and HCP who should communicate the diagnosis to KS children/adolescents. Our results show that most PRE-D mothers and fathers prefer KS diagnosis communication at 14–18 years of age, while most KS patients and POST-D parents indicate that the best timing for KS diagnosis communication is before 14 years of age. Concerning the HCP in charge of diagnosis communication, heterogeneous answers were expressed, suggesting the need for a multidisciplinary team for this purpose. Indeed, there is agreement in the literature on the importance and value of the multidisciplinary team as a means of providing care for KS patients , diagnosis communication during childhood/adolescence is a more delicate process, which could generate frustration and anxiety , all HCPs who do not frequently deal with KS. In a study by Bourke et al. (2013), KS parents reported that HCPs, including GPs, pediatricians, obstetricians, and neurologists, provided information indicating that their knowledge of KS treatment and management was not up-to-date.In our study, PRE-D mothers/fathers and POST-D mothers/fathers received adequate information about fertility, cognitive, and hormonal features (consensus reached); metabolic issues were not fully addressed (NA/ND). In these cases, the diagnosis was communicated mostly by geneticists (for PRE-D parents) and by endocrinologists (for POST-D parents), indicating that these HCPs are more familiar with the various aspects of KS syndrome. The theme of fertility was the principal issue explained, most likely because it is the characteristic sign of KS and clinicians may consider it to be the key feature to communicate. Indeed, Dennis and colleagues showed that the most common parental concerns were related to fertility issues and their impact on a child’s self-esteem . In particular, the role of the psychologist is to address emotional/psychological aspects of KS, whereas clinicians are fundamental for providing the correct health information on KS. Suwannachat et al.  suggesteFinally, our study is the first to investigate what participants thought about emotions generated by sharing genetic conditions with teachers, friends, and schoolmates. POST-D parents and KS patients disagreed that talking about KS elicits fear and pity among the listeners and agreed that the sharing of diagnosis may generate empathy among the listeners (consensus reached). In contrast, PRE-D families neither agreed nor disagreed that sharing the diagnosis could generate misunderstanding, fear, and pity among the listeners and disagreed that it may elicit empathy. Suwannachat et al.  showed tA number of limitations affect our study. The sample size is not large and is composed only of Caucasian subjects with a high level of education and mostly of mothers with prenatal diagnosis. Moreover, there are few fathers and their level of education was high. We have a limited number of answers from KS patients that received diagnosis communication during adolescence/childhood. Survey responses are based only on participant reports and subject to recall bias (participants may have some difficulty remembering). The option “ < 14 years” for passing information to the child may be too broad and so it would be useful to split up this category into different sub-categories  to define better at which age an early disclosure should occur. In future, other questions might be added to our questionnaire aimed at filling in the gap in our understanding; these could include the age at which parents communicated diagnosis to their children in real life, emotions aroused by diagnosis communication  among KS patients, and KS patients’ opinion (not only KS parents’ opinion) about the words to use/not to use during communication. In future researches, a combination of both qualitative and quantitative approach could be used for data collection and it might be useful to ask KS patients whether they would prefer the “seed planting strategy” or the “right time strategy” for diagnosis disclosure  by sharing diagnosis.Our conclusions report that communication should be performed before 14 years of age (according to most KS patients and POST-D mothers/fathers’ answers), whereas both PRE-D fathers and mothers believe that the best timing is between 14 and 18 years of age. However, the general preference is that communication should not occur after 18 years of age. Concerning the HCP in charge of diagnosis communication, the answers were heterogeneous, suggesting the need for a multidisciplinary team for this purpose. KS patients reported they did not receive information about metabolic and cognitive features. Clinicians should, therefore, provide information to KS patients not only on fertility and hormonal aspects but also on the aforementioned issues. Our study shows that POST-D parents and KS patients think that sharing diagnosis beyond the family does not generate fear and pity among listeners but empathy Table .Table 6DSupplementary file1 (DOC 57 KB)Below is the link to the electronic supplementary material.


A woman in her thirties came to our primary eye care centre (vision centre) complaining of foreign body sensation and discharge in her right eye which she had been experiencing for three weeks. She had been bitten in the eye by an ant and an insect particle had been removed from the eye one week before the onset of symptoms. She had applied topical antibiotics that were prescribed locally but there was no improvement.On examination at the vision centre, the vision in the right eye was 20/20. All the anterior segment findings were normal. However, upon eversion of the eyelid, a reddish-pink vascular pedunculated lesion was visible on the palpebral conjunctiva of the upper eyelid of the right eye .The vision technician took a photograph  using thWhat is pyogenic granuloma?A vascular lesion of infectious aetiologyA benign, non-tender, exuberant proliferation of granulomatous tissue in response to trauma or surgeryA malignant vascular proliferative lesionA tender granulomatous lesion of inflammatory aetiologyWhat is the key differential diagnosis for conjunctival pyogenic granuloma?DermolipomaDermoid cystConjunctival concretionsSquamous cell carcinomaWhat are the management options for conjunctival pyogenic granuloma?Topical antibiotics, hot fomentation, lid hygieneTopical corticosteroids, removal of inciting agent, excision biopsy, pulsed dye laserTopical lubricants and lid scrubs with baby shampooTopical anti-inflammatory agents and oral analgesicsb. Pyogenic granuloma is a benign, exuberant proliferation of granulomatous lesions due to angiogenic imbalance as a result of of minor trauma, foreign body, or surgery. Eversion of the eyelid is important in order to correctly diagnose these lesions.d. The term pyogenic granuloma is a misnomer, as the lesion is neither infectious nor inflammatory in nature. It is a benign, non-tender lesion. Differentiating from malignant lesions is important in order to avoid unnecessary treatment. The main difference is the slow growth of malignant lesions in contrast to the rapid growth of pyogenic granulomas.b. Conjunctival pyogenic granulomas of smaller size usually respond to topical corticosteroids or pulsed dye lasers. Pulsed dye lasers are effective and safe for the treatment of small and solitary pyogenic granulomas, treated with a vascular-specific (585 nm), pulsed (450 microseconds) dye laser using a 5 mm spot size with a laser energy of 6 to 7 J/cm2. Many patients will eventually require surgical excision of the granuloma along with the removal of the inciting agent or irritant. Excision biopsy allows histological confirmation, and is helpful if there is any doubt about the diagnosis.


Anaphylactic shock is the severe state of the allergic reaction, which is rapid in onset and fatal. This is the first study that discusses the anaphylactic shock of exenatide reexposure in the patient who has interrupted exenatide treatment.A 47-year-old man was treated with exenatide owing to high blood glucose and obesity. Then he developed localized urticarial on the face, white lip, hands tremble, nausea, vomit, chest stuffiness, dizziness, accompanying with confusion and dyspnea. His blood glucose was 4.6 millimole per liter (mmol/L) and blood pressure was 85/50 millimeters of mercury (mm Hg).Exenatide-induced anaphylactic shock was considered.The emergency electrocardiogram was performed. The patient was treated with dexamethasone sodium phosphate and calcium gluconate, combined with exenatide withdrawal. He also received oral antiallergic agents and intravenous nutrition treatment.After antishock treatment, the clinical response gradually alleviated.Although exenatide is not prone to anaphylaxis, it is the synthetic peptide that can induce antibody formation. Exenatide has immunogenicity with the potential to elicit an allergic reaction upon administration. Clinicians should always pay more attention to the anaphylactic shock of exenatide, when prescribing for diabetics. The common adverse effects of exenatide reported in patients include gastrointestinal disorders, pancreatic damage, hypoglycemia, and injection-site reactions. However, anaphylaxis induced by exenatide is very rare . Anaphylactic shock is the severe form of anaphylaxis, and it is the fast developing and potentially fatal systemic reaction.Exenatide is a glucagon-like peptide-1 receptor agonist, which is the first novel incretin analogues with natural glucagon-like peptide-1 activity for treatment of type 2 diabetes.On March 25, 2016, a 47-year-old man was admitted to hospital owing to high blood glucose and urine protein revealed in physical examination. He had personal history of type 2 diabetes for 14 months and urine protein for one month treated with antidiabetic drugs irregularly. The patient also suffered from high blood pressure for over 4 years with the peak blood pressure at 170/120 millimeters of mercury (mm Hg). He was able to keep his blood pressure at about 140/100 mm Hg with irbesartan and aspirin orally. No history of drug or food allergies was found. The patient has provided informed consent for publication of the case.2 with height 181cm and weight 124 kg. He had been treated with exenatide since the diagnosis of diabetes. The initial dose was 5 μg subcutaneously twice daily (b.i.d.), and the subsequent maintenance dose was 10 μg subcutaneously b.i.d. The patient was discharged after stable control of blood glucose and blood pressure. He continued to take exenatide, but self-discontinued it due to scleroma at the injection site. Without any other treatment, the symptom was relieved, therefore, we failed to be informed. When the patient was admitted again because of diabetes, we administrate the hypoglycemic , antihypertensive  and antiplatelet  agents. On March 29, 2016, exenatide was added at a dose of 10 μg in the morning and 20 μg in the evening due to poor blood glucose control, combining with the severe obesity. At 16:30 on April 5, exenatide was given, and half an hour later, he developed generalized urticarial. The symptom was gradually relieved after administration of loratadine tablets. At 6:40 in the morning of 7 of April, he developed localized urticarial on the face, white lip, hands tremble, nausea, vomit, chest stuffiness, dizziness, accompanying with confusion and dyspnea. His blood glucose was 4.6 mmol/L and blood pressure was 85/50 mm Hg. There were no abnormal findings by emergency electrocardiogram. Dexamethasone sodium phosphate and calcium gluconate were commenced. Upon treatment, his blood glucose was 8.6mmol/L and blood pressure was 120/102 mm Hg. However, He still felt nausea, vomiting, dizziness and discomfort. Because the above symptoms were induced after the injection of exenatide, he received treatment with oral hypoglycemic agents instead of exenatide, accompanying with oral antiallergic agents and intravenous nutrition treatment. The clinical response gradually alleviated and the patient’s mood was calmed down. All these clinical symptoms and examinations provide an assistance for confirming the diagnosis of exenatide-induced anaphylactic shock.The patient had a body mass index of 37.8 kg/mIn our study, the patient suffered urticarial, and sequently anaphylactic shock after exenatide injection. Anaphylactic shock is the severe state of the allergic reaction, which is rapid in onset, and if not handled promptly, sometimes it can prove fatal. Within half an hour after the injection of exenatide, the patient suffered anaphylactic shock which exhibits cardiovascular symptoms such as drop of blood pressure to 85/50 mm Hg, chest tightness, dizziness, and confusion. With respect to the gastrointestinal tract, the patient displayed typical symptoms like nausea and vomiting. The abnormal performance of the anaphylactic reaction was clearly correlated with the injection time of exenatide. The symptoms develop immediately  after exposure to the specific allergen (exenatide). The yinxin damo injection and kudie zi injection had been administrated to the patient while hospitalized. However, no intravenous drug was administered prior to the onset of anaphylaxis induced by exenatide. Moreover, the allergic reactions of oral drugs including metformin hydrochloride sustained release tablets, acarbose tablets, irbesartan tablets, aspirin enteric-coated tablets, shiwei longdan hua particles, and qingyan dropping pill are very rare or not mentioned in the specification. And the administration time was not consistent with occurrence of allergy symptoms, which can basically exclude the interference of these drugs. There were no other abnormalities pre- and post- medication and no exposure to other allergens. Therefore, the patient was treated with an intermittent therapy of exenatide, which was helpful in confirming the diagnosis of exenatide-induced anaphylactic shock. When the patients were treatment by exenatide, anti-exenatide antibodies were more common. Low antibody titers do not appear to impacting the efficacy of exenatide, however, high antibody titers in a small percentage of patients reduced mean efficacy, which was statistically significant in the group with exenatide treatment once a week. Anti-exenatide antibodies also associated with the injection site reactions. Numerous research studies reported that antibody formation may induce several consequences,,6 it is supposed that exenatide has immunogenicity with the potential to elicit an allergic reaction upon administration. We have retrieved all the papers that are published in the pubmed up to now and found that there were only 3 reports referred for the systemic hypersensitivity reactions induced by exenatide in patients. One of them reported anaphylactic reaction in the patient with long acting release exenatide (2 mg once-weekly), namely, generalized urticarial, urticarial, uvular edema and vomiting. The second is a patient that suffered from generalized urticaria, and difficulty swallowing after the administration of exenatide. Although the third study reported an exenatide treatment with no allergic reactions because of well tolerated exenatide, the patient was allergic to exenatide by skin prick test and intradermal skin test, with the probable cause that drug skin test results was not always consistent with the clinical effects. The positive skin tests in all these studies and a basophil activation test with positive results in the first study forcefully supported an immunoglobulin E-mediated mechanism. Moreover, discontinuous treatment in our study may increase the risk of anaphylaxis. This is consistent with the second study that intermittent treatment of exenatide has developed an allergy, the possible cause is immunoglobulin E-mediated degranulation of mast cells. In addition, although off-label dose of exenatide in our study was mentioned, exenatide overdose has no clinical harm.,11Although exenatide is not prone to anaphylaxis, it is the synthetic peptide originally isolated from the Heloderma suspectum lizard, which can induce antibody formation that is commonly present in therapeutic peptides.Although anaphylaxis of exenatide has been reported, this is the first study that discusses the anaphylactic shock of exenatide reexposure in the patient who has interrupted exenatide treatment. Anaphylaxis, even anaphylactic shock of exenatide has not arose as frequent problems to date, which need us to paying more attention to its safety. Although we could not get permission to study the mechanism of anaphylactic shock further, we explore the possible allergic reactions, which can attract more attention of clinicians facilitating taking an effect treatment regimen.Methodology: Xujing Liu, Aihua Zhai.Supervision: Bai Zhang.Writing – original draft: Xujing Liu, Aihua Zhai.