Probes for INS

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.

Infection by SARS-CoV-2 may be associated with testicular dysfunction that could affect male fertility.Testicles of fatal COVID-19 cases were investigated to detect virus in tissue and to evaluate histopathological and transcriptomic changes.Three groups were compared: a. uninfected controls (subjects dying of trauma or sudden cardiac death; n = 10); b. subjects dying of COVID-19 (virus-negative in testes; n = 15); c. subjects dying of COVID-19 (virus-positive in testes; n = 9). SARS-CoV-2 genome and nucleocapsid antigen were probed using RT-PCR, in situ hybridization, immunohistochemistry (IHC). Infiltrating leukocytes were typed by IHC. mRNA transcripts of immune-related and testis-specific genes were quantified using the nCounter method.SARS-CoV-2 was detected in testis tissue of 9/24 (37%) COVID-19 cases accompanied by scattered T-cell and macrophage infiltrates. Size of testicles and counts of spermatogenic cells were not significantly different among groups. Analysis of mRNA transcripts showed that in virus-positive testes immune processes were activated (interferon-alpha and -gamma pathways). By contrast, transcription of 12 testis-specific genes was downregulated, independently of virus positivity in tissue. By IHC, expression of the luteinizing hormone/choriogonadotropin receptor was enhanced in virus-positive compared to virus-negative testicles, while expression of receptors for androgens and the follicle-stimulating hormone were not significantly different among groups.In lethal COVID-19 cases, infection of testicular cells is not uncommon. Viral infection associates with activation of interferon pathways and downregulation of testis-specific genes involved in spermatogenesis. Due to the exceedingly high numbers of infected people in the pandemic, the impact of virus on fertility should be further investigated.

The polyamines putrescine, spermidine and spermine are organic polycations that regulate many cell functions including proliferation and differentiation. It is known that certain genes of the polyamine system are dysregulated after kidney ischemia reperfusion injury. Here we examined the hypothesis that different forms of acute and chronic kidney injury lead to similar changes in the expression patterns of the polyamine system. In different models of acute and chronic kidney injury expression of genes involved in polyamine homeostasis were analyzed by RT-qPCR and RNAScope. In these models, expression of catabolic enzymes (Aoc1 and Sat1) was upregulated, and the anabolic enzymes (Odc1, Sms) were downregulated. The putrescine-degrading enzyme AOC1 exhibits the most striking changes. Interestingly, it can act together with ODC1 as gatekeepers of the polyamine system. The detected increase of Aoc1 takes place in the injured but regenerating proximal tubules. As a screening for stimuli of increased Aoc1 expression, we used mouse embryonic kidney explants. Here we observed changes of Aoc1expression under hypoxia and hyperosmotic conditions. These changes were further examined in mouse models of hypoxia. However, in vivo, hypoxia did not lead to changes of Aoc1 expression. Hyperosmolarity was confirmed as a stimulus by using the kidney cell lines M15 and 209/MDCT as well as cultured primary proximal tubules. Using reporter gene and RNA-stability assays, we could show that the increase in Aoc1 expression is based on mRNA-stabilization and transcriptional activation of one certain isoform. The activated isoform contains an additional set of 22 amino acids N-terminally that lead to an altered subcellular localization. In conclusion, different models of kidney injury exhibit a similar pattern of dysregulation of the polyamine system with the most striking change being the upregulation of Aoc1 in proximal tubules. Using hyperosmolarity as a stimulus, we provide first insights into the regulation of Aoc1 under harmful conditions.

Although anesthesia is commonly used in the fields of medicine and scientific research, the neural mechanisms and circuits through which it produces analgesia is still unclear. Utilizing c-fos labeling of neuronal activity, this project aimed to investigate the brain regions of C57BL/6 mice, which become activated subsequent to isoflurane anesthesia. RNAscope in situ hybridization was used to examine c-fos mRNA activation in the brain. Confocal microscopy was utilized to locate and characterize brain regions displaying c-Fos activation. Finally, manual quantification of c-fos activation in identified brain regions was conducted through Fiji software. The brain regions identified resemble brain areas that have been associated with pain regulation in literature, including the central nucleus of amygdala (CeA), paraventricular nucleus of the hypothalamus (PVN), centrally-projecting Edinger-Westphal nucleus (EWcp), piriform cortex (PC), and para-supraoptic nucleus (ParaSON). Furthermore, the CeA displayed the greatest average number of positive cells and the densest activation, supporting its importance in pain and analgesia. The identified brain regions validate the prominent findings of prior studies, which also found c-Fos activation subsequent to isoflurane anesthesia in the CeA, PVN, and ParaSON (Hua et al., Nat Neurosci, 2020). New regions of c-fos activation, including the EWcp and PC, found in this study are in need of further exploration. PC activation may also be caused by smell from isoflurane. The connections and coordination which the identified brain regions have in producing analgesia is also an area for future investigation. This study is supported by Duke University Anesthesiology Fund and NIH grant R01-DE29342. This study is supported by Duke University Anesthesiology Fund and NIH grant R01-DE29342.

A meta-analysis study was conducted to measure the consequence of diabetic foot ulcers (DFUs) and other risk factors (RFs) on the prevalence of lower extremity amputation (LEA). A comprehensive literature inspection till February 2023 was applied and 2765 interrelated studies were reviewed. Of the 32 chosen studies enclosed, 9934 subjects were in the chosen studies' starting point, and 2906 of them were with LEA. Odds ratio (OR) in addition to 95% confidence intervals (CIs) were used to compute the value of the effect of DFUs and other RFs on the prevalence of LEA by the continuous and dichotomous approaches and a fixed or random effect model. Male gender (OR, 1.30; 95% CI, 1.17-1.44, P < .001), smoking (OR, 1.24; 95% CI, 1.01-1.53, P = .04), previous foot ulcer (OR, 2.69; 95% CI, 1.93-3.74, P < .001), osteomyelitis (OR, 3.87; 95% CI, 2.28-6.57, P < .001), gangrene (OR, 14.45; 95% CI, 7.03-29.72, P < .001), hypertension (OR, 1.17; 95% CI, 1.03-1.33, P = .01), and white blood cells count (WBCC) (MD, 2.05; 95% CI, 1.37-2.74, P < .001) were significantly shown to be an RF in LEA in subjects with DFUs. Age (MD, 0.81; 95% CI, -0.75 to 2.37, P = .31), body mass index (MD, -0.55; 95% CI, -1.15 to 0.05, P = .07), diabetes mellitus type (OR, 0.99; 95% CI, 0.63-1.56, P = .96), and glycated haemoglobin (MD, 0.33; 95% CI, -0.15 to 0.81, P = .17) were not shown to be an RF in LEA in subjects with DFUs. Male gender, smoking, previous foot ulcer, osteomyelitis, gangrene, hypertension, and WBCC were significantly shown to be an RF in LEA in subjects with DFUs. However, age and diabetes mellitus type were not shown to be RF in LEA in subjects with DFUs. However, caused of the small sample sizes of several chosen studies for this meta-analysis, care must be exercised when dealing with its values.

Facioscapulohumeral muscular dystrophy (FSHD) is among the most prevalent muscular dystrophies, ranging from 1 in 8,333 to 1 in 20,000. Currently no treatment exists that alters the course of FSHD, and therapy development remains an unmet need in the field. Abnormal reactivation of the DUX4 gene in skeletal muscle has emerged as an underlying cause of muscle weakness and wasting in FSHD. We propose that DUX4 silencing is the most direct route to FSHD therapy. Toward this goal, we developed an AAV6-CRISPR-Cas13 strategy to silence DUX4 mRNA. Cas13 targets and cleaves RNA instead of DNA, and avoids potential risks of permanent off-target genome editing that could arise with DNA-targeting systems. Intramuscular delivery of an AAV6 vector encoding a PspCas13b enzyme and DUX4-targeting guide RNAs reduced DUX4 mRNA by >50% and improved histopathological outcomes in FSHD mice. To investigate possible off-target effects, we performed RNA-seq of treated versus control or untreated human myoblasts and also examined potential collateral RNA cleavage activity using a dual reporter system. Although we did not detect collateral cleavage, our RNA-sequencing results suggested some guide RNAs could induce potential off-target gene expression changes. We are currently exploring mechanisms to explain these differential off-target effects. To address whether PspCas13b can activate a mammalian host immune response, we injected wild-type mice with AAV-Cas13b and investigated immune cell infiltration and pro-inflammatory cytokine profiles. We find evidence of an immune response against PspCas13b in injected mouse muscles. Importantly, transient immunosuppression reduced immune responses to Cas13b in treated animals. In conclusion, our data support that Cas13b can target and reduce DUX4 expression in FSHD muscles, but minimizing cellular immune response may be necessary to translate AAV-Cas13b therapy.

Rationale DNA damage accumulation is a hallmark of vascular smooth muscle cell (VSMC) ageing. Importantly, VSMC DNA damage accumulation and ageing has been implicated in the progression of cardiovascular disease (CVD), including atherosclerosis and vascular calcification. Chemotherapy drugs used in the treatment of many cancers are known to induce DNA damage in cardiovascular cells and accelerate CVD. Histone deacetylase (HDAC) inhibitors are drugs being investigated for novel treatments of many cancers. HDACs perform many vital functions in cells; HDAC6 is known to deacetylate alpha-tubulin to regulate microtubule stability and flexibility. We have recently shown that microtubule stability regulates both VSMC morphology and contractility. Therefore, in this study we investigate the impact of HDAC6 inhibition upon VSMC function. Methodology We use polyacrylamide hydrogels (PAHs)

We previously showed the importance of TGFβ signaling in development of the mouse axial skeleton. Here, we provide the first direct evidence that TGFβ signaling is required for resegmentation of the sclerotome using chick embryos. Lipophilic fluorescent tracers, DiO and DiD, were microinjected into adjacent somites of embryos treated with or without TGFβRI inhibitors, SB431542, SB525334 or SD208, at developmental day E2.5 (HH16). Lineage tracing of labeled cells was observed over the course of 4 days until the completion of resegmentation at E6.5 (HH32). Vertebrae were malformed and intervertebral discs were small and misshapen in inhibitor injected embryos. Hypaxial myofibers were also increased in thickness after treatment with the inhibitor. Inhibition of TGFβ signaling resulted in alterations in resegmentation that ranged between full, partial, and slanted shifts in distribution of DiO or DiD labeled cells within vertebrae. Patterning of rostro-caudal markers within sclerotome was disrupted at E3.5 after treatment with TGFβRI inhibitor with rostral domains expressing both rostral and caudal markers. We propose that TGFβ signaling regulates rostro-caudal polarity and subsequent resegmentation in sclerotome during spinal column development.

SR-17018 was identified as a highly G protein-biased mu opioid peptide (MOP) receptor agonist and lacked MOP agonist-associated adverse effects in mice. The aim of this study was to determine the functional profile of spinal and systemic administration of SR-17018 in non-human primates. In vivo effects of SR-17018 were compared with those of MOP agonists in different intrinsic efficacies, DAMGO, morphine, heroin, and buprenorphine, in behavioral assays established in rhesus monkeys (Macaca mutatta). Nociceptive, itch-scratching, and operant behaviors were measured by experimenters blinded to the dosing conditions. Following intrathecal delivery, SR-17018 (30-300 ug), buprenorphine (3-10 ug), morphine (10-30 ug), and DAMGO (1-3 ug), dose-dependently attenuated capsaicin-induced thermal allodynia (p < 0.05). However, unlike DAMGO and morphine eliciting robust scratching activities, intrathecal SR-17018 and buprenorphine only elicited mild scratching responses, indicating that SR-17018 has low efficacy for activating spinal MOP receptors. In the intravenous drug self-administration assay, heroin (0.3-10 ug/kg/infusion) produced a higher reinforcing strength (abuse liability) as compared to lower reinforcing strengths by SR-17018 (3-30 ug/kg/infusion) and buprenorphine (1-10 ug/kg/infusion) in primates under the progressive-ratio schedule of reinforcement (p < 0.05). The intrathecal opioid-induced itch and intravenous drug self-administration have been documented to distinguish MOP receptor agonists with different intrinsic efficacies. Our findings reveal that in vivo apparent low efficacy of SR-17018 is similar to that of a MOP partial agonist buprenorphine measured by the primate assays with translation relevance. Such a low intrinsic efficacy explains its improved side-effect profile of a highly G protein-biased MOP agonist, S