BACKGROUND:Acute respiratory distress syndrome (ARDS) causes significant morbidity and mortality during viral pneumonia, including SARS-CoV-2 infections. Nevertheless, most patients with SARS-CoV-2 infections recover seamlessly without developing ARDS, suggesting the existence of endogenous pathways to protect the lungs. Since microRNAs (miRNAs) can regulate endogenous molecular pathways involved in lung protection, we hypothesized that alveolar miRNAs could function to dampen SARS-CoV-2-associated lung injury. METHODS:Screening studies in human alveolar epithelial cells and SARS-CoV-2-infected mice were performed to identify miRNAs induced during infection. Candidate miRNAs were confirmed via RT-qPCR. The role of hypoxia-inducible factor 1A (HIF1A) in regulating miRNA expression was examined in molecular studies. Loss- and gain-of-function approaches in a murine SARS-CoV-2 ARDS model were used to assess the physiological relevance of miRNA. Viral sequence analyses and site-directed mutagenesis were used to determine direct miRNA - viral RNA interactions. RESULTS:Screening studies identified miR-147b (hsa-miR-147b-3p or mmu-miR-147-3p) as the leading candidate during infection of human alveolar epithelia or mice with SARS-CoV-2. Functional and molecular studies implicate HIF1A in miR-147b induction during alveolar injury or SARS-CoV-2 infection. Studies in mice with induced deletion of miR-147b in alveolar epithelia (miR147fl/fl SPC-CreER mice) or nano-particle-mediated miR-147b overexpression revealed a protective role of alveolar-expressed miR-147b during murine SARS-CoV-2-associated ARDS. Moreover, we identified the ORF8 region within the SARS-CoV-2 template strand as a direct target of miR-147b, with an ORF8 silent mutation of the SARS-CoV-2 miR-147b-binding site abolishing the observed protection in vitro and in vivo. CONCLUSIONS:Alveolar epithelial cell-derived miR-147b serves as an endogenous lung protective miRNA against SARS-CoV-2-associated ARDS by directly targeting virus-encoded RNA, revealing a previously unrecognized antiviral and lung-protective mechanism.

Hypoxia-inducible factors (HIFs) promote lung protection and pathogen eradication during acute lung injury. We, therefore, tested the theory that pharmacologic stabilization of HIFs dampens lung injury during SARS-CoV-2 pneumonia. Initial studies in murine SARS-CoV-2 models showed improved outcomes after treatment with the FDA-approved HIF stabilizer vadadustat. Subsequent studies in genetic models implicated alveolus-expressed Hif1a in mediating lung protection. Therefore, we performed a randomized, double-blinded, multicenter phase II trial in patients admitted for SARS-CoV-2 infection and concomitant hypoxia (SpO2 ≤ 94%). Patients (n = 448) were randomized to oral vadadustat (900 mg/day) or placebo for up to 14 days. Safety events were similar between the 2 groups. Vadadustat treatment induced surrogate HIF target genes. The primary outcome of severe lung injury requiring high oxygen support on day 14 occurred in 43 patients in the vadadustat group and 53 patients in the placebo group (estimated probability, 13.3% vs. 16.9%). Among patients with baseline fraction of inspired oxygen of 80% or higher (n = 106), the estimated probability of the primary outcome was 12.1% (vadadustat) versus 79.1% (placebo), indicating an even greater benefit in patients with more severe baseline hypoxia. HIF1A is a likely therapeutic target during SARS-CoV-2-associated lung injury. Robust clinical trials of HIF stabilizers during pathogen-associated lung injury are warranted.

Leishmania major is the etiological agent of cutaneous leishmaniasis (CL) in several countries in Asia and Northern Africa. The disease is considered a zoonotic infection where rodents are the reservoirs and phlebotomine sandflies are the vectors. Once inside the human body, the parasite multiplies inside the macrophages of infected patients, but the disease eventually cures spontaneously, leaving scars where the phlebotomine bites occurred. Given the importance of the replicative forms in the parasite’s cell cycle, here, we decided to study the enzyme telomerase, which has the critical role of replenishing the chromosomal telomeric ends during cell replication. To this aim, we first conducted partial purification using Sephacryl-300 HR gel filtration, which allowed us to determine that the telomerase activity eluted as a 600 KDa complex. Second, we characterized an immunopurified L. major telomerase, and to try to explain some of our findings, we performed modeling studies using Alfa fold 3, Pyre2, and Swiss Protein Model. Finally, considering the similarity between the catalytic site of Leishmania and Homo sapiens telomerase, we decided to test typical inhibitors of human telomerase on the purified enzyme and promastigote cell forms, confirming that MST-312 and TMPYP4 efficiently inhibited L. major activity and arrested cell growth in Leishmania promastigotes. Our findings confirm the importance of telomerase activity in L. major’s replicative forms and suggest the possibility of using drugs previously tested on human telomerase to treat CL.

Acute respiratory distress syndrome (ARDS) causes morbidity and mortality during SARS-CoV-2 infections. Nevertheless, most patients with SARS-CoV-2 infection recover seamlessly without developing ARDS. Here, we hypothesized a functional role of microRNAs (miRNAs) in endogenous lung protection during SARS-CoV-2-associated ARDS. Screening studies identified miR-147b (hsa-miR-147b-3p or mmu-miR-147-3p) as the lead candidate during murine and human SARS-CoV-2 infections. Functional molecular studies implicate hypoxia-inducible factor 1A (HIF1A) in miR-147b induction. Subsequent loss-and-gain-of-function studies revealed a protective role of alveolar-expressed miR-147b during murine SARS-CoV-2-associated ARDS. Proof-of-principle studies in patients also implicate this pathway during SARS-CoV-2 infection. Finally, we identified SARS-CoV-2 ORF8 as a direct miR-147b target, and silent mutation of the viral miR-147b-binding site within ORF-8 abolished the observed protection. Together, our findings identify a previously unrecognized role of miR-147b in attenuating SARS-CoV-2-associated lung disease by targeting the viral genome. R01HL154720, R01DK122796, R01HL133900, R01HL155950, R01HL169519, T32GM135118; Department of Defense Grant W81XWH2110032; Parker B. Francis Fellowship; American Lung Association Catalyst Award CA-622265. Viral Immunology (VIR)

Glomeruli are signal-processing units of the olfactory bulb (OB), playing a key role in many OB computations, including contrast enhancement, gain control, and odorant-selective habituation. In awake mice, we uncover an extremely stable inhomogeneous map of basal glomerulus-specific activity as a background against which olfactory signal processing is performed. This activity is strongly driven by (i) centrifugal cholinergic projections, (ii) endogenous, and (iii) airflow-evoked spiking of olfactory sensory neurons and, to a small extent, (iv) by the odor environment. Importantly, early olfactory impairment in a mouse model of Alzheimer’s disease is parallelled by the loss of glomerular map inhomogeneity and diminished cholinergic innervation. These results reveal an important layer in the signal-processing network of the OB, likely acting by increasing the variance in and dynamic range of the system via glomerulus-specific functional inhomogeneity. Teaser A new layer in the signal-processing network of the olfactory bulb supports olfactory-driven behavior. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, GA 654/16-1, GA 654/18-1

Trypanosoma brucei is the etiologic agent of African trypanosomiasis, also known as sleeping sickness. In the mammalian host, T. brucei is present as replicative and non-replicative bloodstream forms (BSF) referred to as long slender and stumpy, respectively. In the insect vector, the developmental forms are procyclic (PCF), epimastigote, and metacyclic trypomastigote. It is well established that BSF use glucose as the main energy source, and PCF can use glucose but, in its absence, uses amino acids, mainly proline. Due to these facts, the metabolism of amino acids has been well studied in PCF, but less attention has been given to it in BSF. In the present work, we unveil the participation of glutamine (Gln), the most abundant amino acid in the human blood, in BSF proliferation. According to the data presented herein, in the absence of extracellular Gln, cell cycle progress, as well as kinetoplast and flagella distribution and function among daughter cells, are widely compromised, yielding polyploid multiflagellated cells with reduced motility. In addition, the Gln-poor medium resulted in cells with a massive alteration in the proteome glutamylation. These alterations were reverted when Gln was added to the culture media. An analysis of the glutamylproteome of cells incubated in Gln-poor medium, followed by a Gene Ontology (GO) analysis, suggested a relationship between glutamylation and biological processes such as gene expression and regulation, cell cycle, and bioenergetics metabolism, in addition to cytoskeleton dynamics in BSF of T. brucei . ### Competing Interest Statement The authors have declared no competing interest.

Acute myocardial infarction stands as a prominent cause of morbidity and mortality worldwide1-6. Clinical studies have demonstrated that the severity of cardiac injury following myocardial infarction exhibits a circadian pattern, with larger infarct sizes and poorer outcomes in patients experiencing morning onset myocardial infarctions7-14. However, the molecular mechanisms that govern circadian variations of myocardial injury remain unclear. Here, we show that BMAL114-20, a core circadian transcription factor, orchestrates diurnal variability in myocardial injury. Unexpectedly, BMAL1 modulates circadian-dependent cardiac injury by forming a transcriptionally active heterodimer with a non-canonical partner, hypoxia-inducible factor 2 alpha (HIF2A)6,21-23, in a diurnal manner. Substantiating this finding, we determined the cryo-EM structure of the BMAL1/HIF2A/DNA complex, revealing a previously unknown capacity for structural rearrangement within BMAL1, which enables the crosstalk between circadian rhythms and hypoxia signaling. Furthermore, we identified amphiregulin (AREG) as a rhythmic transcriptional target of the BMAL1/HIF2A heterodimer, critical for regulating circadian variations of myocardial injury. Finally, pharmacologically targeting the BMAL1/HIF2A-AREG pathway provides effective cardioprotection, with maximum efficacy when aligned with the pathway's circadian trough. Our findings not only uncover a novel mechanism governing the circadian variations of myocardial injury but also pave the way for innovative circadian-based treatment strategies, potentially shifting current treatment paradigms for myocardial infarction.

BACKGROUND:Key functions of Ca2+ signaling in rodent microglia include monitoring the brain state as well as the surrounding neuronal activity and sensing the danger or damage in their vicinity. Microglial Ca2+ dyshomeostasis is a disease hallmark in many mouse models of neurological disorders but the Ca2+ signal properties of human microglia remain unknown. METHODS:We developed a novel genetically-encoded ratiometric Ca2+ indicator, targeting microglial cells in the freshly resected human tissue, organotypically cultured tissue slices and analyzed in situ ongoing Ca2+ signaling of decades-old microglia dwelling in their native microenvironment. RESULTS:The data revealed marked compartmentalization of Ca2+ signals, with signal properties differing across the compartments and resident morphotypes. The basal Ca2+ levels were low in ramified and high in ameboid microglia. The fraction of cells with ongoing Ca2+ signaling, the fraction and the amplitude of process Ca2+ signals and the duration of somatic Ca2+ signals decreased when moving from ramified via hypertrophic to ameboid microglia. In contrast, the size of active compartments, the fraction and amplitude of somatic Ca2+ signals and the duration of process Ca2+ signals increased along this pathway.

The human respiratory and circulatory systems collaborate intricately to ensure oxygen delivery to all cells, which is vital for ATP production and maintaining physiological functions and structures. During limited oxygen availability, hypoxia-inducible factors (HIFs) are stabilized and play a fundamental role in maintaining cellular processes for hypoxia adaptation. First discovered during investigations of erythropoietin production regulation, HIFs influence physiological and pathological processes, including development, inflammation, wound healing, and cancer. HIFs promote extracellular adenosine signaling by enhancing adenosine generation and receptor signaling, representing an endogenous feedback mechanism that curbs excessive inflammation, supports injury resolution, and enhances hypoxia tolerance. This is especially important for conditions that involve tissue hypoxia, such as acute respiratory distress syndrome (ARDS), which globally poses significant health challenges without specific treatment options. Consequently, pharmacological strategies to amplify HIF-mediated adenosine production and receptor signaling are of great importance.

Introduction:Invasion of the central nervous system (CNS) is the most serious consequence of Trypanosoma brucei infection, which causes sleeping sickness. Recent experimental data have revealed some more insights into the disease during the meningoencephalitic stage. However, detailed cellular processes befalling the CNS during the disease are poorly understood.Methods:To further address this issue, we implanted a cranial window on the cortex of B6.129P2(Cg)-Cx3cr1tm1Litt/J mice, infected them with Trypanosoma brucei expressing RFP via intraperitoneal injection, and monitored microglial cells and parasites longitudinally over 30 days using in vivo 2-photon imaging. We correlated the observed changes with histological analyses to evaluate the recruitment of peripheral immune cells.Results and discussion:We uncovered an early involvement of microglia that precedes invasion of the CNS by the parasite. We accomplished a detailed characterization of the progressive sequence of events that correlates with microglial morphological changes and microgliosis. Our findings unveiled a heterogeneous microglial response in places of initial homeostatic disruption near brain barriers and pointed out an exceptional capability of microglia to hamper parasite proliferation inside the brain. We also found early signs of inflammation in the meninges, which synchronize with the microglial response. Moreover, we observed a massive infiltration of peripheral immune cells into the parenchyma as a signature in the final disease stage. Overall, our study provides new insights into the host-pathogen immune interactions in the meningeal and parenchymal compartments of the neocortex.

The development and survival of adult-born neurons are believed to be driven by sensory signaling. Here, in vivo analyses of motility, morphology and Ca2+ signaling, as well as transcriptome analyses of adult-born juxtaglomerular cells with reduced endogenous excitability (via cell-specific overexpression of either Kv1.2 or Kir2.1 K+ channels), revealed a pronounced impairment of migration, morphogenesis, survival, and functional integration of these cells into the mouse olfactory bulb, accompanied by a reduction in cytosolic Ca2+ fluctuations, phosphorylation of CREB and pCREB-mediated gene expression. Moreover, K+ channel overexpression strongly downregulated genes involved in neuronal migration, differentiation, and morphogenesis and upregulated apoptosis-related genes, thus locking adult-born cells in an immature and vulnerable state. Surprisingly, cells deprived of sensory-driven activity developed normally. Together, the data reveal signaling pathways connecting the endogenous intermittent neuronal activity/Ca2+ fluctuations as well as enhanced Kv1.2/Kir2.1 K+ channel function to migration, maturation, and survival of adult-born neurons.

Trypanosoma brucei is the causative agent of human African trypanosomiasis. The parasite transmigrates from blood vessels across the choroid plexus epithelium to enter the central nervous system, a process that leads to the manifestation of second stage sleeping sickness. Using an in vitro model of the blood-cerebrospinal fluid barrier, we investigated the mechanism of the transmigration process. For this, a monolayer of human choroid plexus papilloma cells was cultivated on a permeable membrane that mimics the basal lamina underlying the choroid plexus epithelial cells. Plexus cells polarize and interconnect forming tight junctions. Deploying different T. brucei brucei strains, we observed that geometry and motility are important for tissue invasion. Using fluorescent microscopy, the parasite's moving was visualized between plexus epithelial cells. The presented model provides a simple tool to screen trypanosome libraries for their ability to infect cerebrospinal fluid or to test the impact of chemical substances on transmigration.

Diffuse gliomas, particularly glioblastomas, are incurable brain tumours 1 . They are characterized by networks of interconnected brain tumour cells that communicate via Ca 2+ transients 2 – 6 . However, the networks’ architecture and communication strategy and how these influence tumour biology remain unknown. Here we describe how glioblastoma cell networks include a small, plastic population of highly active glioblastoma cells that display rhythmic Ca 2+ oscillations and are particularly connected to others. Their autonomous periodic Ca 2+ transients preceded Ca 2+ transients of other network-connected cells, activating the frequency-dependent MAPK and NF-κB pathways. Mathematical network analysis revealed that glioblastoma network topology follows scale-free and small-world properties, with periodic tumour cells frequently located in network hubs. This network design enabled resistance against random damage but was vulnerable to losing its key hubs. Targeting of autonomous rhythmic activity by selective physical ablation of periodic tumour cells or by genetic or pharmacological interference with the potassium channel KCa3.1 (also known as IK1, SK4 or KCNN4) strongly compromised global network communication. This led to a marked reduction of tumour cell viability within the entire network, reduced tumour growth in mice and extended animal survival. The dependency of glioblastoma networks on periodic Ca 2+ activity generates a vulnerability 7 that can be exploited for the development of novel therapies, such as with KCa3.1-inhibiting drugs.

Chagas disease, caused by the kinetoplastid parasite Trypanosoma cruzi, is a human tropical illness mainly present in Latin America. The therapies available against this disease are far from ideal. Proteases from pathogenic protozoan have been considered as good drug target candidates. T. cruzi acidic M17 leucyl-aminopeptidase (TcLAP) mediates the major parasite’s leucyl-aminopeptidase activity and is expressed in all parasite stages. Here, we report the inhibition of TcLAP (IC50 = 66.0 ± 13.5 µM) by the bestatin-like peptidomimetic KBE009. This molecule also inhibited the proliferation of T. cruzi epimastigotes in vitro (EC50 = 28.1 ± 1.9 µM) and showed selectivity for the parasite over human dermal fibroblasts (selectivity index: 4.9). Further insight into the specific effect of KBE009 on T. cruzi was provided by docking simulation using the crystal structure of TcLAP and a modeled human orthologous, hLAP3. The TcLAP-KBE009 complex is more stable than its hLAP3 counterpart. KBE009 adopted a better geometrical shape to fit into the active site of TcLAP than that of hLAP3. The drug-likeness and lead-likeness in silico parameters of KBE009 are satisfactory. Altogether, our results provide an initial insight into KBE009 as a promising starting point compound for the rational design of drugs through further optimization.

Olfaction, or the sense of smell, is one of the most ancient senses in men and mice, important for a large variety of innate and acquired behaviors. Clinical data reveal an early impairment of olfaction during normal aging and in the course of neurodegenerative diseases, but the underlying cellular/molecular mechanisms remain obscure. In the current review, we compare different aspects of the aging- and Alzheimer's disease related impairment of olfaction in men and mice, aiming at the identification of common morbidities and biomarkers, which can be analyzed in detail in the appropriate mouse models. We also identify common, often interdependent (patho)physiological pathways, including but not limited to extracellular amyloid depositions, neuroinflammation, ɛ4 allele of the apolipoprotein E, CNS insulin resistance, and the impairment of adult neurogenesis, to be targeted by basic and clinical research.

Trypanosoma brucei is one of the protozoa parasites that can enter the brain and cause injury associated with toxic effects of parasite-derived molecules or with immune responses against infection. Other protozoa parasites with brain tropism include Toxoplasma, Plasmodium, Amoeba, and, eventually, other Trypano-somatids such as T. cruzi and Leishmania. Together, these parasites affect billions of people worldwide and are responsible for more than 500.000 deaths annually. Factors determining brain tropism, mechanisms of in-vasion as well as processes ongoing inside the brain are not well understood. But, they depend on the par-asite involved. The pathogenesis caused by T. brucei initiates locally in the area of parasite inoculation, soon trypanosomes rich the blood, and the disease enters in the so-called early stage. The pathomecha-nisms in this phase have been described, even mole-cules used to combat the disease are effective during this period. Later, the disease evolves towards a late-stage, characterized by the presence of parasites in the central nervous system (CNS), the so-called meningo-encephalitic stage. This phase of the disease has not been sufficiently examined and remains a matter of investigation. Here, I stress the importance of delve into the study of the neuropathogenesis caused by T. brucei, which will enable the identification of path-ways that may be targeted to overcome parasites that reached the CNS. Finally, I highlight the impact that the application of tools developed in the last years in the field of neuroscience will have on the study of neglect-ed tropical diseases.

Throughout the lifespan, microglia, the primary innate immune cells of the brain, fulfill a plethora of homeostatic as well as active immune defense functions, and their aging-induced dysfunctionality is now considered as a key trigger of aging-related brain disorders. Recent evidence suggests that both organism's sex and age critically impact the functional state of microglia but in vivo determinants of such state(s) remain unclear. Therefore, we analyzed in vivo the sex-specific functional states of microglia in young adult, middle aged and old wild type mice by means of multicolor two-photon imaging, using the microglial Ca2 + signaling and directed process motility as main readouts. Our data revealed the sex-specific differences in microglial Ca2 + signaling at all ages tested, beginning with young adults. Furthermore, for both sexes it showed that during the lifespan the functional state of microglia changes at least twice. Already at middle age the cells are found in the reactive or immune alerted state, characterized by heightened Ca2 + signaling but normal process motility whereas old mice harbor senescent microglia with decreased Ca2 + signaling, and faster but disorganized directed movement of microglial processes. The 6-12 months long caloric restriction (70% of ad libitum food intake) counteracted these aging-induced changes shifting many but not all functional properties of microglia toward a younger phenotype. The improvement of Ca2 + signaling was more pronounced in males. Importantly, even short-term (6-week-long) caloric restriction beginning at old age strongly improved microglial process motility and induced a significant albeit weaker improvement of microglial Ca2 + signaling. Together, these data provide first sex-specific in vivo characterization of functional properties of microglia along the lifespan and identify caloric restriction as a potent, cost-effective, and clinically relevant tool for rejuvenation of microglia.

Involvement of the central nervous system (CNS) is the most severe consequence of some parasitic infections. Protozoal infections comprise a group of diseases that together affect billions of people worldwide and, according to the World Health Organization, are responsible for more than 500000 deaths annually. They include African and American trypanosomiasis, leishmaniasis, malaria, toxoplasmosis, and amoebiasis. Mechanisms underlying invasion of the brain parenchyma by protozoa are not well understood and may depend on parasite nature: a vascular invasion route is most common. Immunosuppression favors parasite invasion into the CNS and therefore the host immune response plays a pivotal role in the development of a neuropathology in these infectious diseases. In the brain, microglia are the resident immune cells active in defense against pathogens that target the CNS. Beside their direct role in innate immunity, they also play a principal role in coordinating the trafficking and recruitment of other immune cells from the periphery to the CNS. Despite their evident involvement in the neuropathology of protozoan infections, little attention has given to microglia–parasite interactions. This review describes the most prominent features of microglial cells and protozoan parasites and summarizes the most recent information regarding the reaction of microglial cells to parasitic infections. We highlight the involvement of the periphery–brain axis and emphasize possible scenarios for microglia–parasite interactions.