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Endometriosis (EM) is defined as the engraftment and proliferation of functional endometrial-like tissue outside the uterine cavity, leading to a chronic inflammatory condition. While the precise etiology of EM remains elusive, recent studies have highlighted the crucial involvement of a dysregulated immune system. The complement system is one of the predominantly altered immune pathways in EM. Owing to its involvement in the process of angiogenesis, here, we have examined the possible role of the first recognition molecule of the complement classical pathway, C1q. C1q plays seminal roles in several physiological and pathological processes independent of complement activation, including tumor growth, placentation, wound healing, and angiogenesis. Gene expression analysis using the publicly available data revealed that C1q is expressed at higher levels in EM lesions compared to their healthy counterparts. Immunohistochemical analysis confirmed the presence of C1q protein, being localized around the blood vessels in the EM lesions. CD68+ macrophages are the likely producer of C1q in the EM lesions since cultured EM cells did not produce C1q in vitro. To explore the underlying reasons for increased C1q expression in EM, we focused on its established pro-angiogenic role. Employing various angiogenesis assays on primary endothelial endometriotic cells, such as migration, proliferation, and tube formation assays, we observed a robust proangiogenic effect induced by C1q on endothelial cells in the context of EM. C1q promoted angiogenesis in endothelial cells isolated from EM lesions (as well as healthy ovary that is also rich in C1q). Interestingly, endothelial cells from EM lesions seem to overexpress the receptor for the globular heads of C1q (gC1qR), a putative C1q receptor. Experiments with siRNA to silence gC1qR resulted in diminished capacity of C1q to perform its angiogenic functions, suggesting that C1q is likely to engage gC1qR in the pathophysiology of EM. gC1qR can be a potential therapeutic target in EM patients that will disrupt C1q-mediated proangiogenic activities in EM.

Abstract Cancer-associated immune dysfunction is a major challenge for effective therapies. The emergence of antibodies targeting tumor cell-surface antigens led to advancements in the treatment of hematopoietic malignancies, particularly blood cancers. Yet their impact is constrained against tumors of hematopoietic origin manifesting in the skin. In this study, we employ a clonality-supervised deep learning methodology to dissect key pathological features implicated in mycosis fungoides, the most common cutaneous T-cell lymphoma. Our investigations unveil the prominence of the IL-32β–major histocompatibility complex (MHC)-I axis as a critical determinant in tumor T-cell immune evasion within the skin microenvironment. In patients’ skin, we find MHC-I to detrimentally impact the functionality of natural killer (NK) cells, diminishing antibody-dependent cellular cytotoxicity and promoting resistance of tumor skin T-cells to cell-surface targeting therapies. Through murine experiments in female mice, we demonstrate that disruption of the MHC-I interaction with NK cell inhibitory Ly49 receptors restores NK cell anti-tumor activity and targeted T-cell lymphoma elimination in vivo. These findings underscore the significance of attenuating the MHC-I-dependent immunosuppressive networks within skin tumors. Overall, our study introduces a strategy to reinvigorate NK cell-mediated anti-tumor responses to overcome treatment resistance to existing cell-surface targeted therapies for skin lymphoma.

Introduction Workplaces can be source of significant stress for employees, leading to a series of mental health problems, such as burnout syndrome. Healthcare professionals and other helping professions are especially vulnerable to work-related stress. Objectives The aim of the present review is to assess available intervention aiming at improving work-related stress symptoms. Methods We conducted a thorough search of relevant articles on PubMed, APA PsycInfo, and Scopus databases, using specific keywords such as “occupational stress,” “stress,” “anxiety,” “depression,” “health personnel,” “health care facilities, manpower and services,” “prevention,” and “control.” Results Although significant methodological heterogeneity has been found among studies, regarding assessment tools, target population, and intervention types, we can still draw some satisfactory results. Healthcare professionals have access to various interventions to manage work-related stress symptoms, which can be classified into three categories: 1) individual cognitive-behavioral therapy approaches, 2) relaxation techniques at the individual level, and 3) organizational-level interventions. Mindfulness techniques, relaxation techniques, emotional freedom techniques, and integrated interventions have demonstrated effectiveness in alleviating work-related stress. Conclusions To prevent work-related stress among healthcare professionals, interventions should be targeted towards specific categories of healthcare workers based on factors such as age, tasks, and patient types. Well-structured and reliable randomized controlled trials focusing on the most promising interventions, such as mindfulness, need to be carried out in larger samples and with a solid methodology in order to confirm these evidences. Disclosure of Interest None Declared

Introduction The time period between the onset of a mental disorder and its first adequate treatment (duration of untreated illness - DUI) influence long-term prognosis and outcome in patients with severe mental disorders. The relationship between DUI and outcome was originally found in people affected by schizophrenia spectrum disorders, however in patients with Obsessive-Compulsive Disorder (OCD) DUI is significantly longer compared with that of patients with other severe mental disorders, such as schizophrenia and bipolar disorder. Objectives Aims of the present study is to assess the impact of DUI on long-term outcomes in OCD patients across published studies. Methods A systematic review was carried out by selecting relevant articles on the topic present in three common on-line databases, such as PubMed, APA PsycInfo, and Scopus, up to June 2023. Results Among included studies, DUI ranged from 7,0±8,5 to 20,9±11,2 years. Patients reporting a longer DUI have a poor long-term outcome, in terms of greater symptom severity and lower level of treatment response, whether pharmacological treatment or psychotherapy or a combination of these two. This is particularly true when the onset of the disease is insidious and subthreshold. However, there are severe early-onset forms of OCD in which the request for help is anticipated due to the severity of the symptoms, the DUI is shorter, but the prognosis is still negative. Conclusions The present review confirms that longer DUI has a negative impact on the long-term outcome of patients with OCD. Furthermore, it is reasonable to hypothesize that cultural factors, such as the perception of the disease and the ability to access treatment, may result in a prolongation of the DUI. All these elements cannot be evaluated in our review due to the paucity of studies on the topic. Future studies could be useful to better understand the causes of a longer DUI, to guide and to promote the dissemination of early interventions with a specific focus on OCD symptoms. Disclosure of Interest None Declared

Discrimination between hematopoietic stem cells and leukemic stem cells remains a major challenge for acute myeloid leukemia immunotherapy. CAR T cells specific for the CD117 antigen can deplete malignant and healthy hematopoietic stem cells before consolidation with allogeneic hematopoietic stem cell transplantation in absence of cytotoxic conditioning. Here we exploit non-viral technology to achieve early termination of CAR T cell activity to prevent incoming graft rejection. Transient expression of an anti-CD117 CAR by mRNA conferred T cells the ability to eliminate CD117+ targets in vitro and in vivo. As an alternative approach, we used a Sleeping Beauty transposon vector for the generation of CAR T cells incorporating an inducible Caspase 9 safety switch. Stable CAR expression was associated with high proportion of T memory stem cells, low levels of exhaustion markers, and potent cellular cytotoxicity. Anti-CD117 CAR T cells mediated depletion of leukemic cells and healthy hematopoietic stem cells in NSG mice reconstituted with human leukemia or CD34+ cord blood cells, respectively, and could be terminated in vivo. The use of a non-viral technology to control CAR T cell pharmacokinetic properties is attractive for a first-in-human study in patients with acute myeloid leukemia prior to hematopoietic stem cell transplantation.

Synthetic mRNA produced by in vitro transcription (ivt mRNA) is the active pharmaceutical ingredient of approved anti-COVID-19 vaccines and of many drugs under development. Such synthetic mRNA typically contains several hundred bases of non-coding “untranslated” regions (UTRs) that are involved in the stabilization and translation of the mRNA. However, UTRs are often complex structures, which may complicate the entire production process. To eliminate this obstacle, we managed to reduce the total amount of nucleotides in the UTRs to only four bases. In this way, we generate minimal ivt mRNA (“minRNA”), which is less complex than the usual optimized ivt mRNAs that are contained, for example, in approved vaccines. We have compared the efficacy of minRNA to common augmented mRNAs (with UTRs of globin genes or those included in licensed vaccines) in vivo and in vitro and could demonstrate equivalent functionalities. Our minimal mRNA design will facilitate the further development and implementation of ivt mRNA-based vaccines and therapies.

The release of nanoplastics (NPs) in the environment is a significant health concern for long-term exposed humans. Although their usage has certainly revolutionized several application fields, at nanometer size, NPs can easily interact at the cellular level, resulting in potential harmful effects. Micro/Nanoplastics (M/NPs) have a demonstrated impact on mammalian endocrine components, such as the thyroid, adrenal gland, testes, and ovaries, while more investigations on prenatal and postnatal exposure are urgently required. The number of literature studies on the NPs’ presence in biological samples is increasing. However, only a few offer a close study on the model environmental NP–immune system interaction exploited by advanced microscopy techniques. The present study highlights substantial morphological and lipid metabolism alterations in human M1 macrophages exposed to labeled polypropylene and polyvinyl chloride nanoparticles (PP and PVC NPs) (20 μg/ml). The results are interpreted by advanced microscopy techniques combined with standard laboratory tests and fluorescence microscopy. We report the accurate detection of polymeric nanoparticles doped with cadmium selenide quantum dots (CdSe-QDs NPs) by following the Se (L line) X-ray fluorescence emission peak at higher sub-cellular resolution, compared to the supportive light fluorescence microscopy. In addition, scanning transmission X-ray microscopy (STXM) imaging successfully revealed morphological changes in NP-exposed macrophages, providing input for Fourier transform infrared (FTIR) spectroscopy analyses, which underlined the chemical modifications in macromolecular components, specifically in lipid response. The present evidence was confirmed by quantifying the lipid droplet (LD) contents in PP and PVC NPs-exposed macrophages (0–100 μg/ml) by Oil Red O staining. Hence, even at experimental NPs' concentrations and incubation time, they do not significantly affect cell viability; they cause an evident lipid metabolism impairment, a hallmark of phagocytosis and oxidative stress.

RNA editing refers to non-transient RNA modifications that occur after transcription and prior to translation by the ribosomes. RNA editing is more widespread in cancer cells than in non-transformed cells and is associated with tumorigenesis of various cancer tissues. However, RNA editing can also generate neo-antigens that expose tumour cells to host immunosurveillance. Global RNA editing in melanoma and its relevance to clinical outcome currently remain poorly characterized. The present study compared RNA editing as well as gene expression in tumour cell lines from melanoma patients of short or long metastasis-free survival, patients relapsing or not after immuno- and targeted therapy and tumours harbouring BRAF or NRAS mutations. Overall, our results showed that NTRK gene expression can be a marker of resistance to BRAF and MEK inhibition and gives some insights of candidate genes as potential biomarkers. In addition, this study revealed an increase in Adenosine-to-Inosine editing in Alu regions and in non-repetitive regions, including the hyperediting of the MOK and DZIP3 genes in relapsed tumour samples during targeted therapy and of the ZBTB11 gene in NRAS mutated melanoma cells. Therefore, RNA editing could be a promising tool for identifying predictive markers, tumour neoantigens and targetable pathways that could help in preventing relapses during immuno- or targeted therapies.

Nanoparticles of different sizes formulated with unmodified RNA and Protamine differentially engage Toll-like Receptors (TLRs) and activate innate immune responses in vitro. Here, we report that similar differential immunostimulation that depends on the nanoparticle sizes is induced in vivo in wild type as well as in humanized mice. In addition, we found that the schedule of injections strongly affects the magnitude of the immune response. Immunostimulating 130 nm nanoparticles composed of RNA and Protamine can promote lung metastasis clearance but provides no control of subcutaneous tumors in a CT26 tumor model. We further enhanced the therapeutic capacity of Protamine-RNA nanoparticles by incorporating chemotherapeutic base analogues in the RNA; we coined these immunochemotherapeutic RNAs (icRNAs). Protamine-icRNA nanoparticles were successful at controlling established subcutaneous CT26 and B16 tumors as well as orthotopic glioblastoma. These data indicate that icRNAs are promising cancer therapies, which warrants their further validation for use in the clinic.

In this study, we revealed a peculiar morphological feature of 50B11 nociceptive sensory neurons in in vitro culture related to the forskolin-induced differentiation of these cells growing upside-down on cover glass supports. Multi-photon non-linear microscopy was applied to monitor increased neurite arborization and elongation. Under live and unstained conditions, second harmonic generation (SHG) microscopy could monitor microtubule organization inside the cells while also correlating with the detection of cellular multi-photon autofluorescence, probably derived from mitochondria metabolites. Although the differentiated cells of each compartment did not differ significantly in tubulin or multi-photon autofluorescence contents, the upturned neurons were more elongated, presenting a higher length/width cellular ratio and longer neurites, indicative of differentiated cells. SHG originating from the axons’ microtubules represented a proper tool to study neurons’ inverted culture in live conditions without exogenous staining. This work represents the first instance of examining neuronal cell lines growing and differentiated in an upside-down orientation, allowing a possible improvement of 50B11 as a model in physiology studies of sensory neurons in peripheric nervous system disease (e.g., Fabry disease, Friedreich ataxia, Charcot–Marie–Tooth, porphyria, type 1 diabetes, Guillain–Barré syndrome in children) and analgesic drug screening.

Protamine is a natural cationic peptide mixture used as a drug for the neutralization of heparin and in formulations of slow-release insulin. In addition, Protamine can be used for the stabilization and delivery of nucleic acids (antisense, small interfering RNA (siRNA), immunostimulatory nucleic acids, plasmid DNA, or messenger RNA) and is therefore included in several compositions that are in clinical development. Notably, when mixed with RNA, protamine spontaneously generates particles in the size range of 20–1000 nm depending on the formulation conditions (concentration of the reagents, ratio, and presence of salts). These particles are being used for vaccination and immuno-stimulation. Several grades of protamine are available, and we compared them in the context of complex formation with messenger RNA (mRNA). We found that the different available protamine preparations largely vary in their composition and capacity to transfect mRNA. Our data point to the source of protamine as an important parameter for the production of therapeutic protamine-based complexes.

Mycosis fungoides (MF) is a type of cutaneous T-cell lymphoma. Chlormethine (CL) is recommended as first-line therapy for MF, with a major purpose to kill tumor cells through DNA alkylation. To study the extent of treatment susceptibility and tumor specificity, we investigated the gene expression of different DNA repair pathways, DNA double-stranded breaks, and tumor cell proliferation of clonal TCR Vβ+ tumor cell populations in cutaneous T-cell lymphoma skin cells on direct exposure to CL. Healthy human T cells were less susceptible to CL exposure than two T-lymphoma cell lines, resulting in higher proportions of viable cells. Interestingly, in T cells from MF lesions, we observed a downregulation of several important DNA repair pathways, even complete silencing of RAD51AP1, FANC1, and BRCA2 involved in homologous recombination repair. In the presence of CL, the double-stranded DNA breaks in malignant MF skin T cells increased significantly as well as the expression of the apoptotic gene CASP3. These data point toward an important effect of targeting CL on MF skin tumor T cells, which support CL use as an early cutaneous lymphoma treatment and can be of synergistic use, especially beneficial in the setting of combination skin-directed therapies for cutaneous T-cell lymphoma.

An improved understanding of an ovary’s structures is highly desirable to support advances in folliculogenesis knowledge and reproductive medicine, with particular attention to fertility preservation options for prepubertal girls with malignant tumors. Although currently the golden standard for structural analysis is provided by combining histological sections, staining, and visible 2D microscopic inspection, synchrotron radiation phase-contrast microtomography is becoming a new challenge for three-dimensional studies at micrometric resolution. To this aim, the proper use of contrast agents can improve the visualization of internal structures in ovary tissues, which normally present a low radiopacity. In this study, we report a comparison of four staining protocols, based on iodine or tungsten containing agents, applied to bovine ovarian tissues fixed in Bouin’s solution. The microtomography (microCT) analyses at two synchrotron facilities under different set-ups were performed at different energies in order to maximize the image contrast. While tungsten-based agents allow large structures to be well identified, Iodine ones better highlight smaller features, especially when acquired above the K-edge energy of the specific metal. Further scans performed at lower energy where the setup was optimized for overall quality and sensitivity from phase-contrast still provided highly resolved visualization of follicular and intrafollicular structures at different maturation stages, independent of the staining protocol. The analyses were complemented by X-ray Fluorescence mapping on 2D sections, showing that the tungsten-based agent has a higher penetration in this type of tissues.

Background: Although X-ray fluorescence microscopy is becoming a widely used technique for single-cell analysis, sample preparation for this microscopy remains one of the main challenges in obtaining optimal conditions for the measurements in the X-ray regime. The information available to researchers on sample treatment is inadequate and unclear, sometimes leading to wasted time and jeopardizing the experiment’s success. Many cell fixation methods have been described, but none of them have been systematically tested and declared the most suitable for synchrotron X-ray microscopy. Methods: The HEC-1-A endometrial cells, human spermatozoa, and human embryonic kidney (HEK-293) cells were fixed with organic solvents and cross-linking methods: 70% ethanol, 3.7%, and 2% paraformaldehyde; in addition, HEK-293 cells were subjected to methanol/ C3H6O treatment and cryofixation. Fixation methods were compared by coupling low-energy X-ray fluorescence with scanning transmission X-ray microscopy and atomic force microscopy. Results: Organic solvents lead to greater dehydration of cells, which has the most significant effect on the distribution and depletion of diffusion elements. Paraformaldehyde provides robust and reproducible data. Finally, the cryofixed cells provide the best morphology and element content results. Conclusion: Although cryofixation seems to be the most appropriate method as it allows for keeping cells closer to physiological conditions, it has some technical limitations. Paraformaldehyde, when used at the average concentration of 3.7%, is also an excellent alternative for X-ray microscopy.

Synthetic mRNA acts as a template for synthesizing proteins, protein fragments, or peptides and now has many pharmaceutical applications.^1,2^ Coronavirus disease 2019 (COVID-19) due to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel zoonotic RNA virus, has resulted in the rapid development of dedicated mRNA vaccines.^3–5^ This rapid response was made possible by using mRNA platforms that already existed for experimental vaccines against other infectious diseases and cancer.^6–12^ Carrier-based mRNA vaccines have been developed using lipid-based delivery, peptide-based delivery, polymer-based delivery, and cationic nano-emulsions, as well as dendritic cells.^13^ The mRNA vaccine leads to the expression of encoded antigens in antigen-presenting cells (APCs), generating both innate and adaptive immune responses. Future developments in mRNA therapy in oncology are expected to include adaptations in the routes of administration and co-delivery of multiple mRNAs with other anti-cancer treatments, such as immune checkpoint inhibitors (ICI), radiotherapy, or chemotherapy. In addition, advances in next-generation sequencing (NGS) technology allow the genome, exome, and transcriptome of a single cancer patient to be deciphered. This new knowledge about the diversity of epitopes in different tumors and corresponding specific T cells has allowed the advancement of personalized cancer treatments.^14^ The article aims to present the rationale for the new therapeutic roles of mRNA vaccines, from COVID-19 and other infections to personalized oncology therapeutics.

In the original article [...]

SARS-CoV-2 infection and the resulting COVID-19 have afflicted millions of people in an ongoing worldwide pandemic. Safe and effective vaccination is needed urgently to protect not only the general population but also vulnerable subjects such as patients with cancer. Currently approved mRNA-based SARS-CoV-2 vaccines seem suitable for patients with cancer based on their mode of action, efficacy, and favorable safety profile reported in the general population. Here, we provide an overview of mRNA-based vaccines including their safety and efficacy. Extrapolating from insights gained from a different preventable viral infection, we review existing data on immunity against influenza A and B vaccines in patients with cancer. Finally, we discuss COVID-19 vaccination in light of the challenges specific to patients with cancer, such as factors that may hinder protective SARS-CoV-2 immune responses in the context of compromised immunity and the use of immune-suppressive or immune-modulating drugs.

Akutni bronhiolitis je najpogostejša okužba spodnjih dihal pri otrocih, mlajših od dveh let. Zdravljenje akutnega bronhiolitisa je podporno. Poleg skrbi za primerno hidracijo je dodatek kisika otrokom s hipoksemijo praktično edini način zdravljenja teh otrok. Vrednost, ki jo dobimo s pulznim oksimetrom, je le posredna meritev dejanske vrednosti kisika v krvi in ne odrazi resnosti bolezni. Z dodatkom kisika sicer povišamo zasičenost hemoglobina s kisikom in tako zmanjšamo hipoksemijo, ne zdravimo pa osnovnega vzroka za njen nastanek. Kljub vsemu pulzna oksimetrija ostaja odločilna preiskava pri odločitvi glede zdravljenja s kisikom, saj z nobenim kliničnim znakom ne moremo natančno oceniti, ali gre pri otroku za hipoksemijo. Študije so pokazale, da pediatri pri oceni resnosti bolezni vse bolj zaupajo vrednosti zasičenosti kisika v krvi (SpO2) kot pa klinični oceni. Odkar je pulzna oksimetrija v uporabi, se je odstotek hospitaliziranih zaradi akutnega bronhiolitisa zvišal za približno 250 %. Smernice za obravnavo otrok z akutnim bronhiolitisom niso enotne glede vrednosti SpO2, ki zahtevajo zdravljenje s kisikom. V prispevku smo jih kritično ovrednotili in predstavili lastne izkušnje glede zdravljenja akutnega bronhiolitisa s kisikom.

Background Cocaine use disorder (CUD) is a global health issue with no effective treatment. Repetitive Transcranial Magnetic Stimulation (rTMS) is a recently proposed therapy for CUD. Methods We conducted a single-center, randomised, sham-controlled, blinded, parallel-group research with patients randomly allocated to rTMS (15 Hz) or Sham group (1:1) using a computerised block randomisation process. We enrolled 62 of 81 CUD patients in two years. Patients were followed for eight weeks after receiving 15 15 Hz rTMS/sham sessions over the left dorsolateral prefrontal cortex (DLPFC) during the first three weeks of the study. We targeted the DLFPC following the 5 cm method. Cocaine lapses in twice a week urine tests were the primary outcome. The secondary outcomes were craving severity, cocaine use pattern, and psychometric assessments. Findings We randomly allocated patients to either an active rTMS group (32 subjects) or a sham treatment group (30 subjects). Thirteen (42%) and twelve (43.3%) of the subjects in rTMS and sham groups, respectively, completed the full trial regimen, displaying a high dropout rate. Ten/30 (33%) of rTMS-treated patients tested negative for cocaine in urine, in contrast to 4/27 of placebo controls (p = 0.18, odd ratio 2.88, CI 0.9–10). The Kaplan-Meier survival curve did not state a significant change between the treated and sham groups in the time of cocaine urine negativisation (p = 0.20). However, the severity of cocaine-related cues mediated craving (VAS peak) was substantially decreased in the rTMS treated group (pConclusions In CUD, rTMS could be a useful tool for lowering cocaine craving and consumption. Trial registration The study number on clinicalTrials.gov is NCT03607591.

Sézary syndrome (SS) is a rare, leukemic type of cutaneous T-cell lymphoma (CTCL), for which extracorporeal photopheresis (ECP) is a first-line therapy. Reliable biomarkers to objectively monitor the response to ECP in patients with SS are missing. We examined the quantitative and qualitative impact of ECP on natural killer (NK) cell activity in SS patients, and especially their functional ability for antibody-dependent cell-mediated cytotoxicity (ADCC). Further, we addressed the question whether the magnitude of the effect on ADCC can be associated with the anti-cancer efficacy of ECP in SS patients. We assessed numbers of NK cells, ADCC activity, and treatment response based on blood tumor staging in a cohort of 13 SS patients (8 women, 5 men) treated with ECP as a first-line therapy. Blood samples were collected before treatment start and after an average of 9 months of uninterrupted ECP treatment. NK cell numbers were reduced in SS patients compared to healthy individuals and showed a tendency of recovery after long-term ECP treatment, independent of the clinical response to treatment. Patients with marginal increase (≤1.5 AU-fold) or lack of increase in ADCC activity failed to respond clinically to treatment, while patients with an increased ADCC activity showed a reduction in blood tumor burden. NK-mediated ADCC is selectively enhanced and might be a mechanism underlying the effect of ECP while in addition it can possibly serve as a reliable biomarker to objectively monitor response to ECP in patients with SS.

Sézary syndrome (SS) is an aggressive leukemic variant of cutaneous T-cell lymphoma (L-CTCL) that arises from malignant clonally derived skin-homing CD4+ T cells. Based on advancements in our understanding of the mechanisms underlying L-CTCL, boosting the suppressed immune response emerges as a promising strategy in SS management. Immune checkpoint inhibitory molecules have already demonstrated efficacy in a wide spectrum of malignancies. Currently, agents targeting the programmed death-1 (PD-1) axis are under evaluation in L-CTCL. Here we investigated the expression of PD-1 and its ligands, PD-L1 and PD-L2 in blood and skin from patients with L-CTCL. We demonstrate that PD-1 expression is markedly increased on tumor T cells compared to non-tumor CD4+ T cells from SS patients and to CD4+ cells from healthy individuals. In contrast, PD-L1 shows decreased expression on tumor T cells, while PD-L2 expression is low without significant differences between these groups. Functional PD-1 blockade in vitro resulted in reduced Th2 phenotype of non-tumor T lymphocytes, but enhanced the proliferation of tumor T cells from SS patients. Our study sheds some light on the PD-1 axis in both peripheral blood and skin compartments in SS patients, which may be relevant for the treatment of L-CTCL with immune checkpoint inhibitor.

Developing a deeper knowledge about the impact of DNA and RNA epigenetic mutations on sperm production and fertilization performance is essential for selecting best quality samples in Assisted Reproductive Technologies (ART). Indeed, sperm RNAs adenine and guanine are likely to be methylated in low quality RNA sperm samples and their study requires the employment of techniques able to isolate high quality nucleic acids. UV resonance Raman spectroscopy represents a valuable tool that is able to monitor peculiar molecular modifications occurring predominantly in nucleic acids, being less sensitive to the presence of other biological compounds. In this work, we used an UV Resonance Raman (UVRR) setup coupled to a synchrotron radiation source tuned at 250 nm, in order to enhance sperm RNAs adenine and guanine vibrational signals, reducing also the impact of a fluorescence background typically occurring at lower energies. Despite that our protocol should be further optimized and further analyses are requested, our results support the concept that UVRR can be applied for setting inexpensive tools to be employed for semen quality assessment in ART.

Port silting is a common and natural process which often causes serious inconveniences for safe navigation and requires expensive dredging operations to keep the port operative. Sediment deposition is closely related to the exchange water between the basin and the surrounding environment; one way to limit deposits is by reducing the flow entering the port. However, this may be in contrast with the need for adequate sediment quality, which in turn is closely related to an appropriate water current. This seems to be particularly important in lagoon environments, where sediments are often polluted, making its disposal more complicated and costly. The present paper investigates the situation of the port of Marano Lagunare (Italy) by means of a bidimensional morphological-hydrodynamic and spectral coupled model. To reduce the sediment input into the port, the closure of a secondary port entrance is usually suggested. However, this work demonstrates that a complete dredging of the secondary port inlet allows for an increase in water circulation or efficiency renewal, which ensures a better oxygenation at the bottom of the canals.

As of September 2021, twenty-one anti-COVID-19 vaccines have been approved in the world. Their utilization will expedite an end to the current pandemic. Besides the usual vaccine formats that include inactivated viruses (eight approved vaccines) and protein-based vaccines (four approved vaccines), three new formats have been validated: recombinant adenovirus (six approved vaccines), DNA (one approved vaccine), and messenger RNA (mRNA, two approved vaccines). The latter was the fastest (authorized in 2020 in the EU, the USA, and Switzerland). Most Western countries have reserved or use the protein vaccines, the adenovirus vaccines, and mRNA vaccines. I describe here the different vaccine formats in the context of COVID-19, detail the three formats that are chiefly reserved or used in Europe, Canada, and the USA, and discuss why the mRNA vaccines appear to be the superior format.

The four LEP collaborations, ALEPH, DELPHI, L3 and OPAL, have searched for the neutral Higgs bosons which are predicted by the Minimal Supersymmetric standard model (MSSM). The data of the four collaborations are statistically combined and examined for their consistency with the background hypothesis and with a possible Higgs boson signal. The combined LEP data show no significant excess of events which would indicate the production of Higgs bosons. The search results are used to set upper bounds on the cross-sections of various Higgs-like event topologies. The results are interpreted within the MSSM in a number of "benchmark" models, including CP-conserving and CP-violating scenarios. These interpretations lead in all cases to large exclusions in the MSSM parameter space. Absolute limits are set on the parameter tan β and, in some scenarios, on the masses of neutral Higgs bosons.

Progress in nanotechnology calls for material probing techniques of high sensitivity and resolution. Such techniques are also used for high-impact studies of nanoscale materials in medicine and biology. Soft X-ray microscopy has been successfully used for investigating complex biological processes occurring at micrometric and sub-micrometric length scales and is one of the most powerful tools in medicine and the life sciences. Here, we present the capabilities of the TwinMic soft X-ray microscopy end-station at the Elettra synchrotron in the context of medical and biological imaging, while we also describe novel uses and developments.

The high resolution of synchrotron cryo-nano tomography can be easily undermined by setup instabilities and sample stage deficiencies such as runout or backlash. At the cost of limiting the sample visibility, especially in the case of bio-specimens, high contrast nano-beads are often added to the solution to provide a set of landmarks for a manual alignment. However, the spatial distribution of these reference points within the sample is difficult to control, resulting in many datasets without a sufficient amount of such critical features for tracking. Fast automatic methods based on tomography consistency are thus desirable, especially for biological samples, where regular, high contrast features can be scarce. Current off-the-shelf implementations of such classes of algorithms are slow if used on a real-world high-resolution dataset. In this paper, we present a fast implementation of a consistency-based alignment algorithm especially tailored to a multi-GPU system. Our implementation is released as open-source.

Efficient vaccination can be achieved by injections of in vitro transcribed mRNA (ivt mRNA) coding for antigens. This vaccine format is particularly versatile and allows the production of individualised vaccines conferring, T-cell immunity against specific cancer mutations. The CDR3 hypervariable regions of immune receptors (T-cell receptor, TCR or B-cell receptor, BCR) in the context of T- or B-cell leukaemia or lymphoma are targetable and specific sequences, similar to cancer mutations. We evaluated the functionality of an mRNA-based vaccine designed to trigger immunity against TCR CDR3 regions in an EL4 T-lymphoma cell line-derived murine in vivo model. Vaccination against the hypervariable TCR regions proved to be a feasible approach and allowed for protection against T-lymphoma, even though immune escape in terms of TCR downregulation paralleled the therapeutic effect. However, analysis of human cutaneous T-cell lymphoma samples indicated that, as is the case in B-lymphomas, the clonotypic receptor may be a driver mutation and is not downregulated upon treatment. Thus, vaccination against TCR CDR3 regions using customised ivt mRNA is a promising immunotherapy method to be explored for the treatment of patients with T-cell lymphomas.

The quantification of T-cell immune responses is crucial for the monitoring of natural and treatment-induced immunity, as well as for the validation of new immunotherapeutic approaches. The present study presents a simple method based on lipofection of synthetic mRNA in mononuclear cells as a method to determine in vitro T-cell responses. We compared several commercially available transfection reagents for their potential to transfect mRNA into human peripheral blood mononuclear cells and murine splenocytes. We also investigated the impact of RNA modifications in improving this method. Our results demonstrate that antigen-specific T-cell immunomonitoring can be easily and quickly performed by simple lipofection of antigen-coding mRNA in complex immune cell populations. Thus, our work discloses a convenient solution for the in vitro monitoring of natural or therapy-induced T-cell immune responses.

Protamine is a natural cationic peptide mixture mostly known as a drug for the neutralization of heparin and as a compound in formulations of slow-release insulin. Protamine is also used for cellular delivery of nucleic acids due to opposite charge-driven coupling. This year marks 60 years since the first use of Protamine as a transfection enhancement agent. Since then, Protamine has been broadly used as a stabilization agent for RNA delivery. It has also been involved in several compositions for RNA-based vaccinations in clinical development. Protamine stabilization of RNA shows double functionality: it not only protects RNA from degradation within biological systems, but also enhances penetration into cells. A Protamine-based RNA delivery system is a flexible and versatile platform that can be adjusted according to therapeutic goals: fused with targeting antibodies for precise delivery, digested into a cell penetrating peptide for better transfection efficiency or not-covalently mixed with functional polymers. This manuscript gives an overview of the strategies employed in protamine-based RNA delivery, including the optimization of the nucleic acid’s stability and translational efficiency, as well as the regulation of its immunostimulatory properties from early studies to recent developments.

The use of in vitro transcribed messenger RNA (ivt mRNA) for vaccination, gene therapy and cell reprograming has become increasingly popular in research and medicine. This method can be used in vitro (transfected in cells) or administered naked or formulated (lipoplexes, polyplexes, and lipopolyplexes that deliver the RNA to specific organs, such as immune structures, the lung or liver) and is designed to be an immunostimulatory or immunosilent agent. This vector contains several functional regions (Cap, 5' untranslated region, open reading frame, 3' untranslated region and poly-A tail) that can all be optimised to generate a highly efficacious ivt mRNA. In this study, we review these aspects and report on the effect of the ivt mRNA purification method on the functionality of this synthetic transient genetic vector.

Basic and translational research in reproductive medicine can provide new insights with the application of scanning probe microscopies, such as atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM). These microscopies, which provide images with spatial resolution well beyond the optical resolution limit, enable users to achieve detailed descriptions of cell topography, inner cellular structure organization, and arrangements of single or cluster membrane proteins. A peculiar characteristic of AFM operating in force spectroscopy mode is its inherent ability to measure the interaction forces between single proteins or cells, and to quantify the mechanical properties (i.e., elasticity, viscoelasticity, and viscosity) of cells and tissues. The knowledge of the cell ultrastructure, the macromolecule organization, the protein dynamics, the investigation of biological interaction forces, and the quantification of biomechanical features can be essential clues for identifying the molecular mechanisms that govern responses in living cells. This review highlights the main findings achieved by the use of AFM and SNOM in assisted reproductive research, such as the description of gamete morphology; the quantification of mechanical properties of gametes; the role of forces in embryo development; the significance of investigating single-molecule interaction forces; the characterization of disorders of the reproductive system; and the visualization of molecular organization. New perspectives of analysis opened up by applying these techniques and the translational impacts on reproductive medicine are discussed.

In the race for a vaccine against SARS-CoV-2, the synthetic mRNA format has been shown to be the fastest one and proved to be safe and highly efficient, even at the very low dose of a few µg per injection. The mRNA vaccines are not new: vaccines that are based on attenuated mRNA viruses, such as Mumps, Measles, and Rubella, immunize by delivering their mRNAs into the cells of the vaccinated individual, who produces the viral proteins that then prime the immune response. Synthetic mRNA in liposomes can be seen as a modern, more refined, and thereby a safer version of those live attenuated RNA viruses. The anti-COVID-19 mRNA vaccine (coding the SARS-CoV-2 spike protein) is the third synthetic RNA therapeutic being approved. It follows the aptamer Macugen® (which neutralizes VEGF) and the siRNA Onpattro® (which destroys the transthyretin-coding mRNA). Remarkably, the 30 µg of mRNA that are contained in the first approved anti-COVID-19 vaccine are sufficient for generating high levels of neutralizing antibodies against the virus in all injected volunteers (including participants over 65 years old). The efficacy and safety data are stunning. The distribution of these vaccines throughout the world will bring a halt to the coronavirus pandemic.

Background: RNA editing is a highly conserved posttranscriptional mechanism that contributes to transcriptome diversity. In mammals, it includes nucleobase deaminations that convert cytidine (C) into uridine (U) and adenosine (A) into inosine (I). Evidence from cancer studies indicates that RNA-editing enzymes promote certain mechanisms of tumorigenesis. On the other hand, recoding editing in mRNA can generate mutations in proteins that can participate in the Major Histocompatibility Complex (MHC) ligandome and can therefore be recognized by the adaptive immune system. Anti-cancer treatment based on the administration of immune checkpoint inhibitors enhance these natural anti-cancer immune responses. Results: Based on RNA-Seq datasets, we evaluated the editome of melanoma cell lines generated from patients pre- and post-immunotherapy with immune checkpoint inhibitors. Our results reveal a differential editing in Arthrobacter luteus (Alu) sequences between samples pre-therapy and relapses during therapy with immune checkpoint inhibitors. Conclusion: These data pave the way towards the development of new diagnostics and therapies targeted to editing that could help in preventing relapses during immunotherapies.

The prism of the Lignano tidal inlet was approximately constant over the last forty years, although the section width has halved. This has led to questions concerning the factors that most influence the tidal prism, and on the applicability of the well-known A–P relationship. A conceptual scheme of the sea–channel–lagoon system has been used to perform a sensitivity analysis of different parameters that characterize both the basin and the inlet cross-section. A 2D hydrodynamic model has been applied to evaluate the prism and compare it to the one derived by a static method, which is the basis of the analytical derivation of the A–P linkage. Three regimes have been found in the prism variability as a function of the basin extension: a linear static regime between prism and basin area; an asymptotic regime in which the prism depends only on the basin bottom friction; and an intermediate one. In addition, the roles of the inlet and channel sizes on the prism value have been investigated. The results, compared to the empirical relationships between the prism and the inlet cross-section, show that a variation in the cross-sectional area does not always corresponds to a change in tidal prism.

This data article contains data related to the research article entitled, “Synchrotron X-ray microscopy reveals early calcium and iron interaction with crocidolite fibers in the lung of exposed mice” [1]. Asbestos fibers disrupt iron homeostasis in the human and mouse lung, leading to the deposition of iron (Fe) onto longer asbestos fibers which forms asbestos bodies (AB) [2]. Similar to Fe, calcium (Ca) is also deposited in the coats of the AB. This article presents data on iron and calcium in the mouse lung after asbestos exposure detected by histochemical evaluation. Keywords: Asbestos, Animal model, Iron, Calcium, Histochemistry, Histopathology

Although being a crucial step for Assisted Reproduction Technologies (ART) success, to date sperm selection is based only on morphology, motility and concentration characteristics. Considering the many possible alterations, there is a great need for analytical approaches allowing more effective sperm selections. The use of Fourier Transform Infrared (FTIR) may represent an interesting possibility, being able to reveal many macromolecular changes in a single measurement in a nondestructive way. As a proof of concept, in this observational study, we used a FTIR approach to reveal features related to sperm quality and chemical changes promoted by in vitro capacitation. We found indication that α-helix content is increased in capacitated sperm, while high percentages of the β-structures seem to correlate to poor-quality spermatozoa. The most interesting observation was related to the lipid composition, when measured as CH2/CH3 vibrations (ratio 2853/2870), which resulted in being strongly influenced by capacitation and well correlated with sperm motility. Interestingly, this ratio is higher than 1 in infertile samples, suggesting that motility is related to sperm membranes stiffness and lipid composition. Although further analyses are requested, our results support the concept that FTIR can be proposed as a new smart diagnostic tool for semen quality assessment in ART.

The current knowledge concerning the connection between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the renin–angiotensin system (RAS) system in the male reproductive apparatus is still limited, so dedicated studies are urgently required. Concerns about the male fertility consequences of SARS-CoV-2 infection have started to emerge, since epidemiologic studies observed that this coronavirus affects male patients more frequently and with increased severity, possibly because of the hormone-regulated expression of angiotensin-converting enzyme 2 (ACE2) receptor. A disturbance in fertility is also expected based on studies of the previous SARS-CoV infection, which targets the same ACE2 receptor when entering the host cells. In addition, bioinformatics analyses reveal the abundant expression of ACE2 receptor in the male reproductive tissues, particularly in the testis. It has been proposed that pharmacological intervention favoring the angiotensin-(1–7)/ACE2/Mas receptor pathway and increasing ACE2 expression and activity could greatly prevent inflammatory lesions in this area. Finally, in laboratories performing assisted reproductive technologies it is recommended that more attention should be paid not only to sperm quality but also to safety aspects. Data about the potential infectivity of seminal fluid are in fact conflicting and do not exclude risks for both personnel and patients. The potential infectivity of SARS-CoV-2 in reproductive male tissues should be strongly considered and further investigated for the proper management of in vitro fertilization procedures.