Probes for INS

Diffuse gliomas continue to be an important problem in neuro-oncology. To solve it, studies have considered the issues of molecular pathogenesis from the intratumoral heterogeneity point. Here, we carried out a comparative dynamic analysis of the different cell populations’ content in diffuse gliomas of different molecular profiles and grades, considering the cell populations’ functional properties and the relationship with patient survival, using flow cytometry, immunofluorescence, multiparametric fluorescent in situ hybridization, polymerase chain reaction, and cultural methods. It was shown that an increase in the IDH-mutant astrocytomas and oligodendrogliomas malignancy is accompanied by an increase in stem cells’ proportion and mesenchymal cell populations’ appearance arising from oligodendrocyte-progenitor-like cells with cell plasticity and cells’ hypoxia response programs’ activation. In glioblastomas, malignancy increase is accompanied by an increase in both stem and definitive cells with mesenchymal differentiation, while proneuronal glioma stem cells are the most likely the source of mesenchymal glioma stem cells, which, in hypoxic conditions, further give rise to mesenchymal-like cells. Clinical confirmation was a mesenchymal-like cell and mesenchymal glioma stem cell number, and the hypoxic and plastic molecular programs’ activation degree had a significant effect on relapse-free and overall survival. In general, we built a multi-vector model of diffuse gliomas’ pathogenetic tracing up to the practical plane.

Background Hyperglycaemia is a common finding in diabetic and non-diabetic patients presenting with ACS and is a powerful predictor of prognosis and mortality. The role of hyperglycaemia in ischemia-reperfusion injury (IRI) is not fully understood, and whether the Sodium Glucose Co-Transporter 1 (SGLT1) plays a role in infarct augmentation, before and/or after reperfusion, remains to be elucidated. However, diabetes clinical trials have shown SGLT inhibition improves cardiovascular outcomes, yet the mechanism is not fully understood. Purpose (1) Characterise the expression of SGLT1 in the myocardium, (2) investigate if SGLT1 is involved in a glucotoxicity injury during IRI, and (3) whether inhibiting SGLT1 with an SGLT inhibitor may reduce infarct size. Methods RT-PCR and in-situ hybridization (RNAScope) combined with Immunoflurescence integrated co detection with different cell marker techniques were used to detect SGLT1 mRNA expression in Sprague-Dawley whole myocardium and isolated primary cardiomyocytes. An Ex-vivo Langendorff ischemia-reperfusion perfusion model was used to study the effect of high glucose (22 mmol) on myocardium at reperfusion and Canagliflozin (CANA) a non-selective SGLT inhibitor (1000 nmol/L to block both the SGLT1 receptor and SGLT 2 receptor and 5 nmol/L to block the SGLT2 receptor only) was introduced following ischaemia at two different concentrations, at reperfusion and its effect on infarct size measured using triphenyltetrazolium chloride (TTC) staining. Results RT-PCR found SGLT1 mRNA is expressed in whole myocardium and in individual cardiac chambers. RNAscope detected SGLT1 mRNA is expressed homogenously within intact myocardium, particularly evident within the vasculature. Importantly, hyperglycaemia (22 mmol) at reperfusion increased infarct size (51.80 ± 3.52% vs 40.80 ± 2.89%; p-value: 0.026) compared to normoglycaemia, low dose CANA (5 nmol/L) did not attenuate infarct size in low glucose or high glucose, whereas high CANA concentration (1μmoL/L) significantly reduced infarct size in high glucose (22 mM) when administered at reperfusion (P value = 0.0047). Conclusion We have shown that SGLT1 is present in the myocardium. Hyperglycaemia appears augment myocardial infarction and inhibition of SGLT1 attenuates this increase.

Pseudo-anodontia consists in the clinical, not radiographic, absence of teeth, due to failure in their eruption. It has been reported as part of an extremely rare syndrome, named GAPO syndrome. Pseudo-hypoparathyroidism type 1a (PHPT-1a) is a rare condition, characterized by resistance to the parathyroid hormone (PTH), as well as to many other hormones, and resulting in hypocalcemia, hyperphosphatemia, and elevated PTH. We report here the case of a 32-year-old woman with a long-standing history of non-treated hypocalcemia, in the context of an undiagnosed PHPT-1a. She had an intellectual disability, showed clinical features of the Albright hereditary osteodystrophy (AHO) and presented signs of multiple hormone resistances. She received treatment for seizures since the age of six. Examination of her mouth revealed a complete absence of teeth. Treatment of hypocalcemia and hormone deficiencies were started only at 29 years of age. Genetic testing demonstrated the presence of a frameshift variant in the GNAS gene in the proband as well as in her mother. A Single Nucleotide Polymorphism (SNP) array analysis failed to demonstrate pathogenic copy number variants (CNVs) but showed several regions with loss of heterozygosity (LOHs) for a final percentage of 1.75%, compatible with a fifth degree of relationship. Clinical exome sequencing (CES) ruled out any damaging variants in all the teeth agenesis-related genes. In conclusion, although we performed an extensive genetic analysis in search of possible additional gene alterations that could explain the presence of the peculiar phenotypic characteristics observed in our patient, we could not find any additional genetic defects. Our results suggest that the association of genetically confirmed PHPT-1a and complete pseudo-anodontia associated with persistent patchy alopecia areata is a new additional nonclassical feature related to the GNAS pathogenic variant.

Mucosa-associated lymphoid tissue (MALT) lymphomas often express IgM and IRTA1 with only a minority demonstrating plasmacytic differentiation. However, like primary cutaneous marginal zone lymphoproliferative disorders (PCMZLPD), thyroid MALT lymphomas (T-MALT-L) frequently show plasmacytic differentiation and IgG positivity. Whether T-MALT-L share other features with PCMZLPD, including frequent IgG4 positivity and infrequent IRTA1 expression, and how IRTA1 staining compares to that in Hashimoto thyroiditis (HT) are unknown. Therefore, the clinicopathologic features of 18 T-MALT-L were assessed, and their IRTA1 expression compared with that in 5 HT cases. All T-MALT-L cases included a B-lymphoid component. Plasmacytic differentiation was present in 15 cases and was extensive in 12. Fourteen cases were IgG+ including 2 IgG4+ (12 κ+, 2 κ−/λ−). One case was IgAλ+. Plasmacytic cells were uniformly CD19+/CD56− but CD138− in 7/15 cases. IRTA1+ cells were present in 16/16 cases, ranging from scattered cells to >50%. They were often concentrated in “MALT ball”-type lymphoepithelial lesions, perifollicular regions, and sometimes in germinal centers. IRTA1 positivity was also present in all HT cases, although it was never very extensive and often had a perifollicular distribution, occasionally with sparse aggregates and positive cells within rare thyroid follicles. Thus, T-MALT-L share some features with PCMZLPD but are more similar to noncutaneous MALT lymphomas, with prominent lymphoepithelial lesions, ubiquitous although variable IRTA1 positivity, and infrequent IgG4 positivity. Plasmacytic differentiation is also common although CD138 loss is frequent and light chain staining may be absent. IRTA1 staining may help in the differential diagnosis with HT, although there is some overlap in staining patterns.

The 27th Scientific Conference of the Society on Neuroimmune Pharmacology (SNIP) in New Delhi, India, on March 15-18, 2023 is a historic summit of experts from around the world. The four day conference provides insights into the latest and most advanced science in the intersecting areas of neuroscience, immunology, pharmacology, and its translational aspects, in particular, HIV and drug abuse. Abstracts are ordered in three major groups: (1) Symposium speakers (S1-S64), (2) Investigator Posters (I1-I18), and (3) Trainee Poster (T1-T28).

Programmed Death Ligand 1 (PD-L1) is crucial in regulating the immunological tolerance in non-small cell lung cancer (NSCLC). Alveolar macrophage (AM)-derived PD-L1 binds to its receptor, PD-1, on surveilling lymphocytes, leading to lymphocyte exhaustion. Increased PD-L1 expression is associated with cigarette smoke (CS)-exposure. However, the PD-L1 role in CS-associated lung diseases associated with NSCLC, such as chronic obstructive pulmonary disease (COPD), is still unclear. In two different cohorts of ever smokers with COPD or NSCLC, and ever and never smoker controls, we evaluated PD-L1 expression: (1) via cutting-edge digital spatial proteomic and transcriptomic profiling (Geomx) of formalin-fixed paraffin-embedded (FFPE) lung tissue sections (n = 19); and (2) via triple immunofluorescence staining of bronchoalveolar lavage (BAL) AMs (n = 83). PD-L1 mRNA expression was also quantified in BAL AMs exposed to CS extract. PD-L1 expression was increased in the bronchiolar wall, parenchyma, and vascular wall from mild-moderate (GOLD 1-2) COPD patients compared to severe-very severe (GOLD 3-4) COPD patients and controls. Within all the COPD patients, PD-L1 protein expression was associated with upregulation of genes involved in tumor progression and downregulation of oncosuppressive genes, and strongly directly correlated with the FEV1% predicted, indicating higher PD-L1 expression in the milder vs. more severe COPD stages. In bronchioles, PD-L1 levels were strongly directly correlated with the number of functionally active AMs. In BAL, we confirmed that AMs from patients with both GOLD 1-2 COPD and NSCLC had the highest and similar, PD-L1 expression levels versus all the other groups, independently from active cigarette smoking. Intriguingly, AMs from patients with more severe COPD had reduced AM PD-L1 expression compared to patients with mild COPD. Acute CS extract stimulation increased PD-L1 mRNA expression only in never-and not in ever-smoker AMs. Lungs from patients with mild COPD and NSCLC are characterized by a similar strong PD-L1 expression signature in bronchioles and functionally active AMs compared to patients with severe COPD and controls. Active smoking does not affect PD-L1 levels. These observations represent a new resource in understanding the innate immune mechanisms underlying the link between COPD and lung cancer onset and progression and pave the way to future studies focused on the mechanisms by which CS promotes tumorigenesis and COPD.

Sex differences occur in the structure and function of the rat cerebral cortex and hippocampus, which can change from the juvenile period through old age. Although the evidence is incomplete, it appears that in at least some portions of the cortex these differences develop due to the rise of ovarian hormones at puberty and are potentially not dependent on the perinatal rise in testosterone, which is essential for sexual differentiation of the hypothalamus and sexual behavior. During aging of female rats, the presence of continued ovarian hormone secretion after cessation of the estrous cycle also influences sex differences in neuroanatomical structure and cognitive behavior, resulting in nullification or reversal of sex differences seen in younger adults. Sex differences can be altered by experience in a stimulating environment during the juvenile/adolescent period, and sex differences in performance even can be affected by the parameters of a task. Thus, broad generalizations about differences such as “spatial ability” are to be avoided. It is clear that to understand how the brain produces behavior, sex and hormones have to be taken into account.

Chronic-pain is a debilitating illness that has become exceedingly widespread with currently limited treatments. Differences in the molecular signature of nociceptors, have been demonstrated between human and the commonly-used mouse model, suggesting functional differences in detection and transmission of noxious-stimuli. Therefore, direct understanding of pain-physiology in humans is required for pain treatment. This could be facilitated by studying humans carrying deleterious genetic mutations affecting pain sensation. The transient receptor potential vanilloid 1 (TRPV1) channel is associated with several body-functions, in particular, noxious-heat detection and inflammatory-pain. Reports of adverse effects in human trials have hinder the clinical development of TRPV1 antagonists as novel pain relievers. Hence, studies on the functional roles of TRPV1, which currently rely mainly on evidences obtained from rodents, should be extended to humans. Here, we examined humans carrying a unique missense mutation in TRPV1, rendering the channel non-functional. The affected individual demonstrated lack of aversion towards capsaicin and elevated heat-pain threshold. Surprisingly, he showed elevated cold-pain threshold and extensive neurogenic inflammatory flare and pain-responses following application of the TRPA1 channel-activator, mustard-oil. Our study provides the first direct evidence for pain-related functional-changes linked to TRPV1 in humans, which is a prime target in the development of novel pain-relievers.

Since the formalization of the Central Dogma of molecular biology, the relevance of RNA in modulating the flow of information from DNA to proteins has been clear. More recently, the discovery of a vast set of non-coding transcripts involved in crucial aspects of cellular biology has renewed the enthusiasm of the RNA community. Moreover, the remarkable impact of RNA therapies in facing the COVID19 pandemics has bolstered interest in the translational opportunities provided by this incredible molecule. For all these reasons, the Italian Society of Biophysics and Molecular Biology (SIBBM) decided to dedicate its 17th yearly meeting, held in June 2022 in Rome, to the many fascinating aspects of RNA biology. More than thirty national and international speakers covered the properties, modes of action and applications of RNA, from its role in the control of development and cell differentiation to its involvement in disease. Here, we summarize the scientific content of the conference, highlighting the take-home message of each presentation, and we stress the directions the community is currently exploring to push forward our comprehension of the RNA World 3.0.

Sustained exposure to a young systemic environment rejuvenates aged organisms and promotes cellular function. However, due to the intrinsic complexity of tissues it remains challenging to pinpoint niche-independent effects of circulating factors on specific cell populations. Here, we describe a method for the encapsulation of human and mouse skeletal muscle progenitors in diffusible polyethersulfone hollow fiber capsules that can be used to profile systemic aging in vivo independent of heterogeneous short-range tissue interactions. We observed that circulating long-range signaling factors in the old systemic environment lead to an activation of Myc and E2F transcription factors, induce senescence, and suppress myogenic differentiation. Importantly, in vitro profiling using young and old serum in 2D culture does not capture all pathways deregulated in encapsulated cells in aged mice. Thus, in vivo transcriptomic profiling using cell encapsulation allows for the characterization of effector pathways of systemic aging with unparalleled accuracy.

The mammalian suprachiasmatic nucleus (SCN) functions as a master circadian pacemaker. In order to examine mechanisms by which it keeps time, entrains to periodic environmental signals (zeitgebers), and regulates subordinate oscillators elsewhere in the brain and in the periphery, a variety of molecular methods have been applied. Multiple label immunocytochemistry and in situ hybridization provide anatomical insights that complement physiological approaches (such as ex vivo electrophysiology and luminometry) widely used to study the SCN.The anatomical methods require interpretation of data gathered from groups of individual animals sacrificed at different time points. This imposes constraints on the design of the experiments that aim to observe changes that occur with circadian phase in free-running conditions. It is essential in such experiments to account for differences in the periods of the subjects. Nevertheless, it is possible to resolve intracellular colocalization and regional expression of functionally important transcripts and/or their peptide products that serve as neuromodulators or neurotransmitters. Armed with these tools and others, understanding of the mechanisms by which the hypothalamic pacemaker regulates circadian function is progressing apace.

Targeting specific opioid receptor types in distinct sensory neurons could lead to safer and more effective treatments against pain. However, the extent to which different DRG neurons that express opioid receptors (MOR, DOR, KOR) innervate distinct organs, and what sensory information is encoded by these neurons, represent long-standing questions in the field. To fill this knowledge gap, we utilized novel knock-in mouse lines in which the DNA recombinases Cre and/or Flp are expressed in opioid receptor-positive DRG neurons. We injected adeno-associated viruses to express tdTomato and analyzed the organization of DRG axon terminals in peripheral tissues using tissue clearing and immunostaining protocols. In hairy skin, we observed circumferential nerve endings around hair follicles that are either MOR+ or DOR+. However, DOR+ circumferential endings were also NFH+ whereas MOR+ circumferential endings were not, suggesting that MOR is expressed by high-threshold mechanoreceptors, while DOR is expressed by low-threshold mechanoreceptors activated by stroking of the skin. In glabrous skin, we found a similar divergent organization, with MOR+ and DOR+ axon terminals co-expressing CRGP and NFH, respectively. In the colon, we observed innervation by both KOR+ and MOR+ axons whereas, in the muscle (soleus) and kidney, we found axons that are either MOR+, DOR+, or KOR+. Remarkably, these MOR+, DOR+, or KOR+ axons innervate different sub-regions within these organs and form distinct nerve-ending structures. Collectively, our findings show that MOR+, DOR+, and KOR+ DRG neurons are expressed in largely non-overlapping DRG neuron types that distinctly innervate tissues and presumably differently contribute to sensory perception. National Institutes of Health grant R01DA044481 New York Stem Cell Foundation.