Cone dysfunction in ARR3-mutation-associated early-onset high myopia: an electrophysiological study.

BACKGROUND: Myopia-26, a Mendelian form of early-onset high-myopia (eoHM) caused by mutations in the X-chromosomal ARR3 gene and predominantly affecting females, curiously, may provide an alternative route of investigation to unveil retinal mechanisms underlying pathological eye growth. We conducted a case-control cross-sectional prospective electrophysiological study in genetically characterized Myopia-26 patients (ARR3 heterozygous symptomatic females) compared with high myopes harboring intact ARR3 alleles and one carrier hemizygous male. RESULTS: Participants were 26 volunteers: 10 healthy control females (E-CTRL, mean age = 31.5 ± 8.8 years), one healthy control male, one carrier male of the mutant ARR3 allele and 14 female eoHM patients (mean age = 27.0 ± 13.1 years) divided in two groups: seven without (M-CTRL) and seven with (MYP-26) genetic alteration in the ARR3 gene. The clinical evaluation included complete eye screening and full-field electroretinograms (ERGs) recorded from both eyes under mydriasis. Spherical equivalent was comparable (mean=-9.55 ± 2.46 and - 10.25 ± 3.22 for M-CTRL and MYP-26, respectively) and best corrected visual acuity (BCVA) was significantly different between M-CTRL and MYP-26 (1.0 vs. 0.406 ± 0.253, respectively). E-CTRL and M-CTRL showed similar light-adapted flash and flicker ERG amplitudes; however, the prior values were reduced by ~ 35% (a- and b-waves alike), the latter by ~ 55% in the MYP-26 group (F(2, 45) > 21.821, p < 0.00001). Dark-adapted a-wave amplitudes were slightly reduced (by ~ 20%) in all myopic patients compared to E-CTRL, irrespective of the ARR3 genotype (E-CTRL vs. eoHM, p = 0.038). CONCLUSIONS: The cone dysfunction observed in Myopia-26 patients is specifically linked to the mutation of ARR3, and is not the consequence of eoHM, i.e. elongation of the eye. It may play a role in myopic refractive error development through a yet unconfirmed pathomechanism.

High prevalence of long COVID in anti-TPO positive euthyroid individuals with strongly elevated SARS-CoV-2-specific T cell responses and moderately raised anti-spike IgG levels 23 months post-infection.

Introduction: Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection, the causative agent of coronavirus disease 2019 (COVID-19), causes post-acute infection syndrome in a surprisingly large number of cases worldwide. This condition, also known as long COVID or post-acute sequelae of COVID-19, is characterized by extremely complex symptoms and pathology. There is a growing consensus that this condition is a consequence of virus-induced immune activation and the inflammatory cascade, with its prolonged duration caused by a persistent virus reservoir. Methods: In this cross-sectional study, we analyzed the SARS-CoV-2-specific T cell response against the spike, nucleocapsid, and membrane proteins, as well as the levels of spike-specific IgG antibodies in 51 healthcare workers, categorized into long COVID or convalescent control groups based on the presence or absence of post-acute symptoms. Additionally, we compared the levels of autoantibodies previously identified during acute or critical COVID-19, including anti-dsDNA, anti-cardiolipin, anti-ß2-glycoprotein I, anti-neutrophil cytoplasmic antibodies, and anti-thyroid peroxidase (anti-TPO). Furthermore, we analyzed the antibody levels targeting six nuclear antigens within the ENA-6 S panel, as positivity for certain anti-nuclear antibodies has recently been shown to associate not only with acute COVID-19 but also with long COVID. Finally, we examined the frequency of diabetes in both groups. Our investigations were conducted at an average of 18.2 months (convalescent control group) and 23.1 months (long COVID group) after confirmed acute COVID-19 infection, and an average of 21 months after booster vaccination. Results: Our results showed significant differences between the two groups regarding the occurrence of acute infection relative to administering the individual vaccine doses, the frequency of acute symptoms, and the T cell response against all structural SARS-CoV-2 proteins. A statistical association was observed between the incidence of long COVID symptoms and highly elevated anti-TPO antibodies based on Pearson's chi-squared test. Although patients with long COVID showed moderately elevated anti-SARS-CoV-2 spike IgG serum antibody levels compared to control participants, and further differences were found regarding the positivity for anti-nuclear antibodies, anti-dsDNA, and HbA1c levels between the two groups, these differences were not statistically significant. Disscussion: This study highlights the need for close monitoring of long COVID development in patients with elevated anti-TPO titers, which can be indicated by strongly elevated SARS-CoV-2-specific T cell response and moderately raised anti-spike IgG levels even long after the acute infection. However, our results do not exclude the possibility of new-onset thyroid autoimmunity after COVID-19, and further investigations are required to clarify the etiological link between highly elevated anti-TPO titers and long COVID.

Anti-Inflammatory Cytokine Profiles in Thrombotic Thrombocytopenic Purpura-Differences Compared to COVID-19.

Thromboinflammation/immunothrombosis plays a role in several diseases including thrombotic thrombocytopenic purpura (TTP) and COVID-19. Unlike the extensive research that has been conducted on COVID-19 cytokine storms, the baseline and acute phase cytokine profiles of TTP are poorly characterized. Moreover, we compared the cytokine profiles of TTP and COVID-19 to identify the disease-specific/general characteristics of thromboinflammation/immunothrombosis. Plasma concentrations of 33 soluble mediators (SMs: cytokines, chemokines, soluble receptors, and growth factors) were measured by multiplex bead-based LEGENDplex™ immunoassay from 32 COVID-19 patients (32 non-vaccinated patients in three severity groups), 32 TTP patients (remission/acute phase pairs of 16 patients), and 15 control samples. Mainly, the levels of innate immunity-related SMs changed in both diseases. In TTP, ten SMs decreased in both remission and acute phases compared to the control, one decreased, and two increased only in the acute phase compared to remission, indicating mostly anti-inflammatory changes. In COVID-19, ten pro-inflammatory SMs increased, whereas one decreased with increasing severity compared to the control. In severe COVID-19, sixteen SMs exceeded acute TTP levels, with only one higher in TTP. PCA identified CXCL10, IL-1RA, and VEGF as the main discriminators among their cytokine profiles. The innate immune response is altered in both diseases. The cytokine profile of TTP suggests a distinct pathomechanism from COVID-19 and supports referring to TTP as thromboinflammatory rather than immunothrombotic, emphasizing thrombosis over inflammation as the driving force of the acute phase.

Dipeptide metabolite, glutamyl-glutamate mediates microbe-host interaction to boost spermatogenesis.

The decrease in sperm count and infertility is a global issue that remains unresolved. By screening environmental bacterial isolates, we have found that a novel lactic acid bacterium, Lactiplantibacillus plantarum SNI3, increased testis size, testosterone levels, sperm count, sexual activity and fertility in mice that have consumed the bacteria for four weeks. The abundance of L. plantarum in the colon microbiome was positively associated with sperm count. Fecal microbiota transplantation (FMT) from L. plantarum SNI3-dosed mice improved testicular functions in microbiome-attenuated recipient animals. To identify mediators that confer pro-reproductive effects on the host, untargeted in situ mass spectrometry metabolomics was performed on testis samples of L. plantarum SNI3-treated and control mice. Enrichment pathway analysis revealed several perturbed metabolic pathways in the testis of treated mice. Within the testis, a dipeptide, glutamyl-glutamate (GluGlu) was the most upregulated metabolite following L. plantarum SNI3 administration. To validate the pro-reproductive feature of GluGlu, systemic and local injections of the dipeptide have been performed. γ-GluGlu increased sperm count but had no effect on testosterone. These findings highlight the role of γ-GluGlu in mediating spermatogenetic effects of L. plantarum on the male mouse host and -following relevant human clinical trials- may provide future tools for treating certain forms of male infertility.

LINE-1 ORF1p is a Promising Biomarker in Cervical Intraepithelial Neoplasia Degree Assessment.

Cervical intraepithelial neoplasia (CIN) represents a spectrum of preinvasive squamous lesions within the cervical epithelium, whose identification is a diagnostic challenge due to subtle histomorphological differences among its categories. This study explores ORF1p, a nucleic acid-binding protein derived from long interspersed nuclear element-1 (LINE-1), as a potential biomarker for enhancing CIN diagnosis. A comprehensive analysis of 143 cervical specimens, encompassing CIN I (n=20), CIN II (n=46), CIN III (n=14), invasive cancer (n=32), and nondysplastic cases (normal cervical epithelia (n=24) and atrophy (n=7) were conducted. ORF1p, Ki67, and p16 expressions were evaluated using immunohistochemistry. ORF1p immunopositivity was detected in the vast majority [110/112 (98.2%)] of dysplastic and neoplastic (CIN and invasive cancer) specimens, whereas 19/24 (79.2%) of normal cervical specimens lacked ORF1p expression. The observed pattern of ORF1p expression showed a progressively increasing extent and intensity with advancing CIN grades. CIN I exhibited mild ORF1p expression in the lower one or two-thirds of the cervical epithelium [14/16 (87.5%)], whereas CIN II demonstrated moderate to strong ORF1p expression spanning the lower two-thirds [29/46 (63.0%)]. Pronounced transepithelial ORF1p immunopositivity characterized CIN III cases [13/14 (92.8%)] and cervical cancer [30/32 (93.8%)]. These findings propose ORF1p as a valuable indicator even for detecting CIN I, effectively discerning them from normal cervical tissue (p < 0.0001). Our findings underscore the potential of ORF1p as an early diagnostic marker for cervical neoplasia.

Glass bead system to study mycotoxin production of Aspergillus spp. on corn and rice starches.

Mycotoxin production by aflatoxin B1 (AFB1) -producing Aspergillus flavus Zt41 and sterigmatocystin (ST) -hyperproducer Aspergillus creber 2663 mold strains on corn and rice starch, both of high purity and nearly identical amylose-amylopectin composition, as the only source of carbon, was studied. Scanning electron microscopy revealed average starch particle sizes of 4.54 ± 0.635 µm and 10.9 ± 2.78 µm, corresponding to surface area to volume ratios of 127 1/µm for rice starch and 0.49 1/µm for corn starch. Thus, a 2.5-fold difference in particle size correlated to a larger, 259-fold difference in surface area. To allow starch, a water-absorbing powder, to be used as a sole food source for Aspergillus strains, a special glass bead system was applied. AFB1 production of A. flavus Zt41 was determined to be 437.6 ± 128.4 ng/g and 90.0 ± 44.8 ng/g on rice and corn starch, respectively, while corresponding ST production levels by A. creber 2663 were 72.8 ± 10.0 µg/g and 26.8 ± 11.6 µg/g, indicating 3-fivefold higher mycotoxin levels on rice starch than on corn starch as sole carbon and energy sources. KEY POINTS: • A glass bead system ensuring the flow of air when studying powders was developed. • AFB1 and ST production of A. flavus and A. creber on rice and corn starches were studied. • 3-fivefold higher mycotoxin levels on rice starch than on corn starch were detected.

Comparison of the exploratory behaviour of wild and laboratory mouse species.

In this study, we compared the exploratory behaviour of mound-building mice (Mus spicilegus) and house mice (Mus musculus) with domesticated laboratory mouse strains (BALB/c and C57BL/6). The animals spent 15 minutes in the furnished test box before the exit to the outside world became free. During the 5-minute test, it was noted whether the animal left the familiar environment; if it did, it was recorded in how many seconds. Based on our results, the wild mouse species were more likely to leave the familiar mouse box and explore the outside environment earlier than the laboratory mice. We also found a difference within the wild mouse species, the mound-building mouse being the one that explored the external environment to a greater extent and faster. The effect of domestication manifests in the fact that laboratory mouse strains are less likely to leave their familiar environment and are significantly less active than their wild ancestors.

The Impact of Transportation on the Cortisol Level of Dwarf Rabbits Bred to Animal-Assisted Interventions.

(1) Background: the popularity of rabbits has increased during the last decade and become the third most common companion animal in the EU. Rabbits' participation in Animal-Assisted Interventions (AAIs) is growing. It is highly important to ensure the well-being of the animals in AAIs. Whereas the needs and the advantages of people involved in AAI are becoming more and more evident, the needs of animals are not clearly defined, therefore, it is a great field of inquiry. Animals who are used for AAI need to be transported regularly, which itself might be a source of stress. (2) Methods: the stress of rabbits-caused by transportation-was measured in a non-invasive way: cortisol levels were determined from feces, based on their breakdown products. Eighteen animals were involved in the study. Rabbits experienced a 30 min transportation every second day for two weeks (altogether six times) while 126 samples were collected. (3) Results: rabbits could handle the transportation procedure the first time but subsequently the stress hormone metabolites in feces samples increased regardless of the offered treatments (hay, carrot and apple) during the carriage. (4) Conclusions: those owners who use rabbits for Animal-Assisted Interventions need to take into account that transportation itself is a stressful experience for the animals.

Secondary infections and long-term outcomes among hospitalized elderly and non-elderly patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and treated with baricitinib: a comparative study from the national centre of Hungary.

Baricitinib is considered a first-line treatment for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected adult patients with an associated cytokine storm syndrome (CSS). Our objective was to compare rates of secondary infections and long-term outcomes of elderly and non-elderly patients who received baricitinib for COVID-19. We conducted a single-centre observational study between November 2020 and September 2023, focusing on hospitalized adult SARS-CoV-2 patients with CSS, categorized as elderly (≥ 65 years) and non-elderly (< 65 years). Enrolment, severity stratification, and diagnosis of infectious complications followed predefined criteria. Outcomes of all-cause mortality and rates of non-severe and severe secondary infections were assessed at 1-year post-treatment initiation. Kaplan-Meier analysis was performed for survival analysis. In total, 490 patients were enrolled (median age 65 ± 23 (21-100) years (years, median ± IQR, min-max); 49.18% elderly; 59.59% male). Elderly patients were admitted to the hospital significantly earlier (7 ± 5 days vs. 8 ± 4 days; p = 0.02), experienced a higher occurrence of severe COVID-19 (121/241, 50.21% vs. 98/249, 39.36%; p = 0.02), and required the use of non-invasive ventilation at baseline (167/225, 74.22% vs. 153/236, 64.83%; p = 0.03). At 1 year, all-cause mortality was significantly higher in the elderly subgroup (111/241, 46.06% vs. 29/249, 11.65%; p < 0.01). At 90 days and 1 year, rates of any severe secondary infection were also more prevalent among the elderly (56/241, 23.24% vs. 37/249 14.86%; p = 0.02 and 58/241, 24.07% vs. 39/249, 15.66%; p = 0.02). In conclusion, elderly SARS-CoV-2-infected patients experience a more severe clinical course, higher secondary infection rates, and increased risk for long-term mortality, regardless of immunomodulatory therapy.

Housing European Ground Squirrels (Spermophilus citellus) for an Ex Situ Conservation Program.

European ground squirrel (Spermophilus citellus) populations have declined precipitously over the last 70 years. Its protection cannot be ensured solely by protecting its habitat; it is also necessary to protect the animals ex situ. In our study, within a European ground squirrel species protection program, we examined two elements of indoor housing technology. Knowledge of the animals' needs is essential for captive housing and breeding success, so in our tests, the animals could freely choose both nest-building materials and feed. In the nest material preference test, the animals could choose from three materials with different structures: paper, Lignocel and hay. In the feed preference test, the animals could also choose from three types of feed: commercial rabbit feed, complete rabbit feed and a natural feed mixture. The first two feeds were in granulated format, and the third was a grain feed mix. Among the nesting materials, they preferred hay, which allowed them to build better-quality nests. Among the feeds, they preferred the grain feed mix, the composition closest to their natural feed, and it was the only one that contained animal protein. Our results contribute to the successful maintenance and breeding the European ground squirrel in captivity.

Population Subdivision and Migration Assessment of Mangalica Pig Breeds Based on Pedigree Analysis.

In conserving the genetic diversity of domestic animal breeds, strategies that emphasise between-breed diversity may not be optimal, as they neglect within-breed variation. The aim of the present study was to assess the extent of population subdivision in three Mangalica pig breeds and the contribution of migration to their substructure. Wright's FST coefficient was calculated based on genealogical data with breeding animals born between 1981 and 2023, with three colour variants (Blonde, Swallow-Belly and Red). These Wright's FST coefficients were analysed using multidimensional scaling to reveal the population substructure. The average FST coefficient was 0.04 for the Blonde breed and 0.047 for the Swallow-Belly and Red Mangalica breeds, while these parameters were lower in the active herds at 0.03 and 0.04, respectively. The migration of individuals between herds was 61.63% for the Blonde breed and 75.53% and 63.64% for the Swallow-Belly and Red Magalica breeds, respectively. No population substructure was observed in any of the Mangalica breeds, which can be explained by the extensive migration between herds.

IGH::NSD2 Fusion Gene Transcript as Measurable Residual Disease Marker in Multiple Myeloma.

Multiple myeloma (MM) is the second most common hematological malignancy. Approximately 15% of MM patients are affected by the t(4;14) translocation resulting in the IGH::NSD2 fusion transcript. Breakage occurs in three major breakpoint regions within the NSD2 gene (MB4-1, MB4-2, and MB4-3), where MB4-1 leads to the production of full-length protein, while truncated proteins are expressed in the other two cases. Measurable residual disease (MRD) has been conclusively established as a crucial prognostic factor in MM. The IGH::NSD2 fusion transcript can serve as a sensitive MRD marker. Using bone marrow (BM) and peripheral blood (PB) samples from 111 patients, we developed a highly sensitive quantitative real-time PCR (qPCR) and digital PCR (dPCR) system capable of detecting fusion mRNAs with a sensitivity of up to 1:100,000. PB samples exhibited sensitivity three orders of magnitude lower compared to BM samples. Patients with an MB4-2 breakpoint demonstrated significantly reduced overall survival (p = 0.003). Our novel method offers a simple and sensitive means for detecting MRD in a substantial proportion of MM patients. Monitoring may be carried out even from PB samples. The literature lacks consensus regarding survival outcomes among patients with different NSD2 breakpoints. Our data align with previous findings indicating that patients with the MB4-2 breakpoint type tend to exhibit unfavorable overall survival.

Effective virus-specific T-cell therapy for high-risk SARS-CoV-2 infections in hematopoietic stem cell transplant recipients: initial case studies and literature review.

The COVID-19 pandemic has exacerbated mortality rates among immunocompromised patients, accentuating the need for novel, targeted therapies. Transplant recipients, with their inherent immune vulnerabilities, represent a subgroup at significantly heightened risk. Current conventional therapies often demonstrate limited effectiveness in these patients, calling for innovative treatment approaches. In immunocompromised transplant recipients, several viral infections have been successfully treated by adoptive transfer of virus-specific T-cells (VST). This paper details the successful application of SARS-CoV-2-specific memory T-cell therapy, produced by an interferon-γ cytokine capture system (CliniMACS® Prodigy device), in three stem cell transplant recipients diagnosed with COVID-19 (case 1: alpha variant, cases 2 and 3: delta variants). These patients exhibited persistent SARS-CoV-2 PCR positivity accompanied by bilateral pulmonary infiltrates and demonstrated only partial response to standard treatments. Remarkably, all three patients recovered and achieved viral clearance within 3 to 9 weeks post-VST treatment. Laboratory follow-up investigations identified an increase in SARS-CoV-2-specific T-cells in two of the cases. A robust anti-SARS-CoV-2 S (S1/S2) IgG serological response was also recorded, albeit with varying titers. The induction of memory T-cells within the CD4 + compartment was confirmed, and previously elevated interleukin-6 (IL-6) and IL-8 levels normalized post-VST therapy. The treatment was well tolerated with no observed adverse effects. While the need for specialized equipment and costs associated with VST therapy present potential challenges, the limited treatment options currently available for COVID-19 within the allogeneic stem cell transplant population, combined with the risk posed by emerging SARS-CoV-2 mutations, underscore the potential of VST therapy in future clinical practice. This therapeutic approach may be particularly beneficial for elderly patients with multiple comorbidities and weakened immune systems.

Waning of SARS-CoV-2 Vaccine Effectiveness in COPD Patients: Lessons from the Delta Variant.

Although the COVID-19 pandemic is profoundly changing, data on the effect of vaccination and duration of protection against infection and severe disease can still be advantageous, especially for patients with COPD, who are more vulnerable to respiratory infections. The Hungarian COVID-19 registry was retrospectively investigated for risk of infection and hospitalization by time since the last vaccination, and vaccine effectiveness (VE) was calculated in adults with COPD diagnosis and an exact-matched control group during the Delta variant of concern (VOC) wave in Hungary (September-December 2021). For the matching, sex, age, major co-morbidities, vaccination status, and prior infection data were obtained on 23 August 2021. The study population included 373,962 cases divided into COPD patients (age: 66.67 ± 12.66) and a 1:1 matched group (age: 66.73 ± 12.67). In both groups, the female/male ratio was 52.2:47.7, respectively. Among the unvaccinated, there was no difference between groups in risk for infection or hospitalization. Regarding vaccinated cases, in the COPD group, a slightly faster decline in effectiveness was noted for hospitalization prevention, although in both groups, the vaccine lost its significant effect between 215 and 240 days after the last dose of vaccination. Based on a time-stratified multivariate Cox analysis of the vaccinated cases, the hazard was constantly higher in the COPD group, with an HR of 1.09 (95%: 1.05-1.14) for infection and 1.87 (95% CI: 1.59-2.19) for hospitalization. In our study, COPD patients displayed lower vaccine effectiveness against SARS-CoV-2 infection and hospitalization but a similar waning trajectory, as vaccines lost their preventive effect after 215 days. These data emphasize revaccination measures in the COPD patient population.

CRISPR/Cas9 Mutagenesis through Introducing a Nanoparticle Complex Made of a Cationic Polymer and Nucleic Acids into Maize Protoplasts.

Presently, targeted gene mutagenesis attracts increasing attention both in plant research and crop improvement. In these approaches, successes are largely dependent on the efficiency of the delivery of gene editing components into plant cells. Here, we report the optimization of the cationic polymer poly(2-hydroxypropylene imine) (PHPI)-mediated delivery of plasmid DNAs, or single-stranded oligonucleotides labelled with Cyanine3 (Cy3) or 6-Carboxyfluorescein (6-FAM)-fluorescent dyes into maize protoplasts. Co-delivery of the GFP-expressing plasmid and the Cy3-conjugated oligonucleotides has resulted in the cytoplasmic and nuclear accumulation of the green fluorescent protein and a preferential nuclear localization of oligonucleotides. We show the application of nanoparticle complexes, i.e., "polyplexes" that comprise cationic polymers and nucleic acids, for CRISPR/Cas9 editing of maize cells. Knocking out the functional EGFP gene in transgenic maize protoplasts was achieved through the co-delivery of plasmids encoding components of the editing factors Cas9 (pFGC-pcoCas9) and gRNA (pZmU3-gRNA) after complexing with a cationic polymer (PHPI). Several edited microcalli were identified based on the lack of a GFP fluorescence signal. Multi-base and single-base deletions in the EGFP gene were confirmed using Sanger sequencing. The presented results support the use of the PHPI cationic polymer in plant protoplast-mediated genome editing approaches.

Corrigendum: Methylome changes in Lolium perenne associated with long-term colonisation by the endophytic fungus Epichloë sp. LpTG-3 strain AR37.

[This corrects the article DOI: 10.3389/fpls.2023.1258100.].

Methylome changes in Lolium perenne associated with long-term colonisation by the endophytic fungus Epichloë sp. LpTG-3 strain AR37.

Epichloë spp. often form mutualistic interactions with cool-season grasses, such as Lolium perenne. However, the molecular mechanisms underlying this interaction remain poorly understood. In this study, we employed reduced representation bisulfite sequencing method (epiGBS) to investigate the impact of the Epichloë sp. LpTG-3 strain AR37 on the methylome of L. perenne across multiple grass generations and under drought stress conditions. Our results showed that the presence of the endophyte leads to a decrease in DNA methylation across genomic features, with differentially methylated regions primarily located in intergenic regions and CHH contexts. The presence of the endophyte was consistently associated with hypomethylation in plants across generations. This research sheds new light on the molecular mechanisms governing the mutualistic interaction between Epichloë sp. LpTG-3 strain AR37 and L. perenne. It underscores the role of methylation changes associated with endophyte infection and suggests that the observed global DNA hypomethylation in L. perenne may be influenced by factors such as the duration of the endophyte-plant association and the accumulation of genetic and epigenetic changes over time.

Clinicopathological analysis of diffuse large B-cell lymphoma using molecular biomarkers: a retrospective analysis from 7 Hungarian centers.

Background: The clinical and genetic heterogeneity of diffuse large B-cell lymphoma (DLBCL) presents distinct challenges in predicting response to therapy and overall prognosis. The main objective of this study was to assess the application of the immunohistochemistry- and interphase fluorescence in situ hybridization (FISH)-based molecular markers in the diagnosis of DLBCL and its prognostic value in patients treated with rituximab-based immunochemotherapy. Methods: This is a multicenter, retrospective study, which analyzed data from 7 Hungarian hematology centers. Eligible patients were adults, had a histologically confirmed diagnosis of DLBCL, were treated with rituximab-based immunochemotherapy in the first line, and had available clinicopathological data including International Prognostic Index (IPI). On the specimens, immunohistochemistry and FISH methods were performed. Germinal center B-cell like (GCB) and non-GCB subtypes were classified by the Hans algorithm. Outcomes included overall survival (OS), event-free survival (EFS), and EFS at 2 years (EFS24). For survival analysis, we used Kaplan-Meier curves with the log-rank test and multivariate Cox regression. Results: A total of 247 DLBCL cases were included. Cases were positive for MYC, BCL2, BCL6, and MUM1 expression in 52.1%, 66.2%, 72.6%, and 77.8%, respectively. BCL6 translocation, BCL2 gene copy number (GCN) gain, IGH::MYC translocation, MYC GCN gain, IGH::BCL2 translocation, and BCL6 GCN gain were detected in 21.4%, 14.1%, 7.3%, 1.8%, 7.3%, and 0.9%, respectively. At a median follow-up of 52 months, 140 patients (56.7%) had disease progression or relapse. The Kaplan-Meier estimate for EFS24 was 56.2% (CI: 50.4-62.8%). In univariate analysis, only IPI and BCL6 expression were significant predictors of both OS and EFS, whereas MUM1 predicted EFS only. In multivariate analysis, the IPI score was a significant independent negative, whereas MIB-1 and BCL6 protein expressions were significant independent positive predictors of both OS and EFS. Conclusion: In our study, we found that only IPI, BCL6 protein expression and MIB-1 protein expression are independent predictors of survival outcomes in DLBCL. We did not find any difference in survival by GCB vs. non-GCB subtypes. These findings may improve prognostication in DLBCL and can contribute to designing further research in the area.

Biophysical characterization of the cetacean morbillivirus haemagglutinin glycoprotein.

Cetacean morbillivirus (CeMV) is an enveloped, non-segmented, negative-stranded RNA virus that infects marine mammals, spreading across species and causing lethal disease outbreaks worldwide. Among the eight proteins encoded by the CeMV genome, the haemagglutinin (H) glycoprotein is responsible for the virus attachment to host cell receptors. CeMV H represents an attractive target for antiviral and diagnostic research, yet the elucidation of the molecular mechanisms underlying its role in infection and inter-species transmission was hampered thus far due to the unavailability of recombinant versions of the protein. Here we present the cloning, expression and purification of a recombinant CeMV H ectodomain (rH-ecto), providing an initial characterization of its biophysical and structural properties. Sodium dodecyl sulphate - polyacrylamide gel electrophoresis (PAGE) combined to Western blot analysis and periodic acid Schiff assay showed that CeMV rH-ecto is purifiable at homogeneity from insect cells as a secreted, soluble and glycosylated protein. Miniaturized differential scanning fluorimetry, Blue Native PAGE and size exclusion chromatography coupled to multiangle light scattering revealed that CeMV rH-ecto is globularly folded, thermally stable and exists in solution in the oligomeric states of dimer and multiple of dimers. Furthermore, negative stain electron microscopy single particle analysis allowed us to delineate a low-resolution molecular architecture of the CeMV rH-ecto dimer, which recapitulates native assemblies from other morbilliviral H proteins, such as those from measles virus and canine distemper virus. This set of experiments by orthogonal techniques validates the CeMV rH-ecto as an experimental model for future biochemical studies on its structure and functions.