Evidence for a quadruplex structure in the polymorphic hs1.2 enhancer of the immunoglobulin heavy chain 3’ regulatory regions and its conservation in mammals

Regulatory regions in the genome can act through a variety of mechanisms that range from the occurrence of histone modifications to the presence of protein-binding loci for self-annealing sequences. The final result is often the induction of a conformational change of the DNA double helix, which alters the accessibility of a region to transcription factors and consequently gene expression. A similar to 300 kb regulatory region on chromosome 14 at the 3' end (3'RR) of immunoglobulin (Ig) heavy-chain genes shows very peculiar features, conserved in mammals, including enhancers and transcription factor binding sites. In primates, the 3'RR is present in two copies, both having a central enhancer named hs1.2. We previously demonstrated the association between different hs1.2 alleles and Ig plasma levels in immunopathology. Here, we present the analysis of a putative G-quadruplex structure (tetraplex) consensus site embedded in a variable number tandem repeat (one to four copies) of hs1.2 that is a distinctive element among the enhancer alleles, and an investigation of its three-dimensional structure using bioinformatics and spectroscopic approaches. We suggest that both the role of the enhancer and the alternative effect of the hs1.2 alleles may be achieved through their peculiar three-dimensional-conformational rearrangement

Analysis of Four New Enterococcus faecalis Phages and Modeling of a Hyaluronidase Catalytic Domain from Saphexavirus

Background: Phage therapy (PT), as a method to treat bacterial infections, needs identification of bacteriophages targeting specific pathogenic host. Enterococcus faecalis, a Gram-positive coccus resident in the human gastrointestinal tract, may become pathogenic in hospitalized patients showing acquired resistance to vancomycin and thus representing a possible target for PT. Materials and Methods: We isolated four phages that infect E. faecalis and characterized them by host range screening, transmission electron microscopy, and genome sequencing. We also identified and three-dimensional modeled a new hyaluronidase enzyme. Results: The four phages belong to Siphoviridae family: three Efquatrovirus (namely vB_EfaS_TV51, vB_EfaS_TV54, and vB_EfaS_TV217) and one Saphexavirus (vB_EfaS_TV16). All of them are compatible with lytic cycle. vB_EfaS_TV16 moreover presents a gene encoding for a hyaluronidase enzyme. Conclusions: The identified phages show features suggesting their useful application in PT, particularly the Saphexavirus that may be of enhanced relevance in PT because of its potential biofilm-digestion capability

Evolutive emergence and divergence of an Ig regulatory node: An environmental sensor getting cues from the aryl hydrocarbon receptor?

One gene, the immunoglobulin heavy chain (IgH) gene, is responsible for the expression of all the different antibody isotypes. Transcriptional regulation of the IgH gene is complex and involves several regulatory elements including a large element at the 3’ end of the IgH gene locus (3’RR). Animal models have demonstrated an essential role of the 3’RR in the ability of B cells to express high affinity antibodies and to express different antibody classes. Additionally, environmental chemicals such as aryl hydrocarbon receptor (AhR) ligands modulate mouse 3’RR activity that mirrors the effects of these chemicals on antibody production and immunocompetence in mouse models. Although first discovered as a mediator of the toxicity induced by the high affinity ligand 2,3,7,8-tetracholordibenzo-p-dioxin (dioxin), understanding of the AhR has expanded to a physiological role in preserving homeostasis and maintaining immunocompetence. We posit that the AhR also plays a role in human antibody production and that the 3’RR is not only an IgH regulatory node but also an environmental sensor receiving signals through intrinsic and extrinsic pathways, including the AhR. This review will 1) highlight the emerging role of the AhR as a key transducer between environmental signals and altered immune function; 2) examine the current state of knowledge regarding IgH gene regulation and the role of the AhR in modulation of Ig production; 3) describe the evolution of the IgH gene that resulted in species and population differences; and 4) explore the evidence supporting the environmental sensing capacity of the 3’RR and the AhR as a transducer of these cues. This review will also underscore the need for studies focused on human models due to the premise that understanding genetic differences in the human population and the signaling pathways that converge at the 3’RR will provide valuable insight into individual sensitivities to environmental factors and antibody-mediated disease conditions, including emerging infections such as SARS-CoV-2

The Genetic Landscape of Serbian Populations through Mitochondrial DNA Sequencing and Non-Recombining Region of the Y Chromosome Microsatellites

The Balkan Peninsula is known to represent a complex cultural mosaic and it is a strategic area because it represents a gateway into Europe from the Near East. This research seeks to evaluate the variability of both uniparental markers (mtDNA and non-recombining region of the Y chromosome) to dissect the genetic makeup of Serbians. The whole sample pertains to 257 Serbians (87 from the central region and 170 from the southern area) who have been analyzed for both uniparental genetic markers. The results showed that the extant inhabitants of the Balkan Peninsula have a homogeneous genetic background, despite their linguistic and cultural differences. The obtained data were compared with those of neighboring populations to detect possible relationships among groups. On the whole, the genetic variability of the Balkan populations seems to be due to an admixture process of European and Asian lineages in different proportions whose contributions constitute the current maternal and paternal genetic landscape

Exploratory Study on Chemosensory Event-Related Potentials in Long COVID-19 and Mild Cognitive Impairment: A Common Pathway?

People affected by the Long COVID-19 (LC) syndrome often show clinical manifestations that are similar to those observed in patients with mild cognitive impairments (MCI), such as olfactory dysfunction (OD), brain fog, and cognitive and attentional diseases. This study aimed to investigate the chemosensory-evoked related potentials (CSERP) in LC and MCI to understand if there is a common pathway for the similarity of symptoms associated with these disorders. Eighteen LC patients (mean age 53; s.d. = 7), 12 patients diagnosed with MCI (mean age 67; s.d. = 6), and 10 healthy control subjects (mean age 66; s.d. = 5, 7) were recruited for this exploratory study. All of them performed a chemosensory event-related potentials (CSERP) task with the administration of trigeminal stimulations (e.g., the odorants cinnamaldehyde and eucalyptus). Study results highlighted that MCI and LC showed reduced N1 amplitude, particularly in the left frontoparietal network, involved in working memory and attentional deficits, and a reduction of P3 latency in LC. This study lays the foundations for evaluating aspects of LC as a process that could trigger long-term functional alterations, and CSERPs could be considered valid biomarkers for assessing the progress of OD and an indicator of other impairments (e.g., attentional and cognitive impairments), as they occur in MCI

HS1,2 Ig Enhancer Alleles Association to AIDS Progression in a Pediatric Cohort Infected with a Monophyletic HIV-Strain

Alteration in the humoral immune response has been observed during HIV infection. The polymorphisms of enhancer HS1,2, member of the 3 regulatory region of the Ig heavy chain cluster, may play a role in the variation of the humoral response leading to pathological conditions. To assess the role of the HS1,2 polymorphic variants in the progression of AIDS, the HS1,2-A allelic frequencies were investigated in a cohort of HIV infected pediatric subjects from a nosocomial outbreak with a monophyletic strain of HIV. From a total group of 418 HIV infected children in the outbreak cohort, 42 nonprogressors and 31 progressors without bias due to antiretroviral therapy were evaluated. HS1,2 allele * 1 has been associated with nonprogressors (allelic frequency: 51.19% versus 33.87% in progressors, OR 0.5, and = 0.0437), while allele * 2 has been associated with progression (allelic frequency: 48.39% versus 30.95% in nonprogressors, OR 2.1, and = 0.0393). Further, only subjects carrying allele * 2 in absence of allele * 1, either in homozygous condition for allele * 2 [nonprogressors 2/42 (4.76%), Progressors 7/31 (22.58%), OR 5.8, and = 0.0315] or in combination with other allelic variants [nonprogressors 7/42 (16.67%), Progressors 13/31 (41.93%), OR 3.61, and = 0.0321], have been associated with HIV progression to AIDS. In conclusion, while the HS1,2 allele * 1 has a protective effect on HIV progression when present, allele * 2 is associated with progression toward AIDS when allele * 1 is absent

Position and sequence conservation in Amniota of polymorphic enhancer HS1.2 within the palindrome of IgH 3'Regulatory Region

<p>Abstract</p> <p>Background</p> <p>The Immunoglobulin heavy chain (IgH) 3' Regulatory Region (3'RR), located at the 3' of the constant alpha gene, plays a crucial role in immunoglobulin production. In humans, there are 2 copies of the 3'RR, each composed of 4 main elements: 3 enhancers and a 20 bp tandem repeat. The single mouse 3'RR differs from the two human ones for the presence of 4 more regulative elements with the double copy of one enhancer at the border of a palindromic region.</p> <p>Results</p> <p>We compared the 3'RR organization in genomes of vertebrates to depict the evolutionary history of the region and highlight its shared features. We found that in the 8 species in which the whole region was included in a fully assembled contig (mouse, rat, dog, rabbit, panda, orangutan, chimpanzee, and human), the shared elements showed synteny and a highly conserved sequence, thus suggesting a strong evolutionary constraint. In these species, the wide 3'RR (~30 kb in human) bears a large palindromic sequence, consisting in two ~3 kb complementary branches spaced by a ~3 kb sequence always including the HS1.2 enhancer. In mouse and rat, HS3 is involved by the palindrome so that one copy of the enhancer is present on each side. A second relevant feature of our present work concerns human polymorphism of the HS1.2 enhancer, associated to immune diseases in our species. We detected a similar polymorphism in all the studied Catarrhini (a primate parvorder). The polymorphism consists of multiple copies of a 40 bp element up to 12 in chimpanzees, 8 in baboons, 6 in macaque, 5 in gibbons, 4 in humans and orangutan, separated by stretches of Cytosine. We show specific binding of this element to nuclear factors.</p> <p>Conclusions</p> <p>The nucleotide sequence of the palindrome is not conserved among evolutionary distant species, suggesting pressures for the maintenance of two self-matching regions driving a three-dimensional structure despite of the inter-specific divergence at sequence level. The information about the conservation of the palindromic structure and the settling in primates of the polymorphic feature of HS1.2 show the relevance of these structures in the control and modulation of the Ig production through the formation of possible three-dimensional structures.</p

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Microbiota and Phage Therapy: Future Challenges in Medicine

An imbalance of bacterial quantity and quality of gut microbiota has been linked to several pathologies. New strategies of microbiota manipulation have been developed such as fecal microbiota transplantation (FMT); the use of pre/probiotics; an appropriate diet; and phage therapy. The presence of bacteriophages has been largely underestimated and their presence is a relevant component for the microbiome equilibrium. As a promising treatment, phage therapy has been extensively used in Eastern Europe to reduce pathogenic bacteria and has arisen as a new method to modulate microbiota diversity. Phages have been selected and &ldquo;trained&rdquo; to infect a wide spectrum of bacteria or tailored to infect specific antibiotic resistant bacteria present in patients. The new development of genetically modified phages may be an efficient tool to treat the gut microbiota dysbiosis associated with different pathologies and increased production of bacterial metabolites and subsequently decrease systemic low-grade chronic inflammation associated with chronic diseases. Microbiota quality and mitochondria dynamics can be remodulated and manipulated by phages to restore the equilibrium and homeostasis of the system. Our aim is to highlight the great interest for phages not only to eliminate and control pathogenic bacterial infection but also in the near future to modulate the microbiota by adding new functions to selected bacteria species and rebalance the dynamic among phages and bacteria. The challenge for the medicine of tomorrow is to re-think and redesign strategies differently and far from our traditional thinking

Low-dose systemic thrombolysis in patient with recent major abdominal surgery and high-risk pulmonary embolism: Case report and narrative review

Introduction: Systemic thrombolysis is the treatment of choice for hemodynamically unstable patients with acute pulmonary embolism (PE) while for the intermediate-high risk ones the possibility of severe bleeding seems to outweigh the benefits. Surgical or percutaneous therapy is an option in case of contraindication/failure of fibrinolysis but they are not available in every hospital. Case Report: An 85-year-old woman arrived at the Emergency Department for rapid onset dyspnea, thoracic pain with hypotension, tachycardia, and hypoxia. A computed tomography pulmonary angiography revealed acute PE of the main branches of the pulmonary right and left arteries. Due to hemodynamic instability and a history of recent intestinal resection surgery (25 days before), a continuous infusion of low-dose thrombolysis regimen with 50 mg rt-PA in 2 hours was started. After 30 minutes of rt-PA infusion, all vital signs were improved and at the end of rt-PA infusion. Point of care ultrasound (POCUS) revealed a reduced size of right ventricle with a decrease in right/left ventricle ratio. Hospital stay and a follow-up at two weeks revealed no complication due to the fibrinolytic administration or anticoagulant therapy. Conclusion: Anticoagulation is still the cornerstone therapy for patients with PE, but there’s a lack of trials on reperfusion strategy. Moreover, different guidelines give different short-term risk classifications, indication, and dosage of thrombolytic drugs and absolute and relative contraindications to systemic thrombolysis. This case report shows the potential benefit of low-dose rt-PA in patients with relative contraindications to thrombolysis and presenting with vital signs compatible with hemodynamic instability