A thorough understanding of complex spatial host-disease interactions _in situ_ is necessary in order to develop effective preventative measures and therapeutic strategies. Here, we developed Protein And Nucleic acid IN situ Imaging (PANINI) and coupled it with Multiplexed Ion Beam Imaging (MIBI) to sensitively and simultaneously quantify DNA, RNA, and protein levels within the microenvironments of tissue compartments. The PANINI-MIBI approach was used to measure over 30 parameters simultaneously across large sections of archival lymphoid tissues from non-human primates that were healthy or infected with simian immunodeficiency virus (SIV), a model that accurately recapitulates human immunodeficiency virus infection (HIV). This enabled multiplexed dissection of cellular phenotypes, functional markers, viral DNA integration events, and viral RNA transcripts as resulting from viral infection. The results demonstrated immune coordination from an unexpected upregulation of IL10 in B cells in response to SIV infection that correlated with macrophage M2 polarization, thus conditioning a potential immunosuppressive environment that allows for viral production. This multiplexed imaging strategy also allowed characterization of the coordinated microenvironment around latently or actively infected cells to provide mechanistic insights into the process of viral latency. The spatial multi-modal framework presented here is applicable to deciphering tissue responses in other infectious diseases and tumor biology.

In spite of the fact that the modulatory effects of angiotensin II (Ang II) on the sympathetic nerve activity to targeted organs involved in blood pressure (BP) regulation is well acknowledged, the local production of this peptide in the brain and the consequences of enhanced central Ang II beyond the cardiovascular system are not yet well comprehended. In the present study, we generated and validated a new transgenic mouse line overexpressing the rat full-length angiotensinogen (Agt) protein specifically in the brain (Agt-Tg). Adult Agt-Tg mice presented overall increased gene expression of total Agt in the brain including brainstem and hypothalamus. In addition, the excess of Agt led to abundantly detectable brain Ang II levels as well as increased circulating copeptin levels. Agt-Tg displayed raised BP in acute recordings, while long-term telemetrically measured basal BP was indistinguishable from wild-types. Agt-Tg has altered peripheral renin-angiotensin system and vasomotor sympathetic tone homeostasis because renal gene expression analysis, plasma Ang II measurements and ganglionic blockade experiments revealed suppressed renin expression and reduced Ang II and higher neurogenic pressure response, respectively. Plasma and urine screens revealed apparently normal fluid and electrolyte handling in Agt-Tg. Interestingly, hematological analyses showed increased hematocrit in Agt-Tg caused by enhanced erythropoiesis, which was reverted by submitting the transgenic mice to a long-term peripheral sympathectomy protocol. Collectively, our findings suggest that Agt-Tg is a valuable tool to study not only brain Ang II formation and its modulatory effects on cardiovascular homeostasis but also its role in erythropoiesis control via autonomic modulation.

Active responses to stressors involve motor planning, execution, and feedback. Here we identify an insular cortex to BNST (insula→BNST) circuit recruited during restraint stress-induced active struggling that modulates affective behavior. We demonstrate that activity in this circuit tightly follows struggling behavioral events and that the size of the fluorescent sensor transient reports the duration of the struggle event, an effect that fades with repeated exposure to the homotypic stressor. Struggle events are associated with enhanced glutamatergic- and decreased GABAergic signaling in the insular cortex, indicating the involvement of a larger circuit. We delineate the afferent network for this pathway, identifying substantial input from motor- and premotor cortex, somatosensory cortex, and the amygdala. To begin to dissect these incoming signals, we examine the motor cortex input, and show that the cells projecting from motor regions to insular cortex are engaged shortly before struggle event onset. This study thus demonstrates a role for the insula→BNST pathway in monitoring struggling activity and regulating affective behavior.

TDP-43 nuclear depletion and concurrent cytoplasmic accumulation in vulnerable neurons is a hallmark feature of progressive neurodegenerative proteinopathies such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Cellular stress signalling and stress granule dynamics are now recognized to play a role in ALS/FTD pathogenesis. Defective stress granule assembly is associated with increased cellular vulnerability and death. Ras-GAP SH3-domain-binding protein 1 (G3BP1) is a critical stress granule assembly factor. Here, we define that TDP-43 stabilizes G3BP1 transcripts via direct binding of a highly conserved cis regulatory element within the 3'UTR. Moreover, we show in vitro and in vivo that nuclear TDP-43 depletion is sufficient to reduce G3BP1 protein levels. Finally, we establish that G3BP1 transcripts are reduced in ALS/FTD patient neurons bearing TDP-43 cytoplasmic inclusions/nuclear depletion. Thus, our data suggest that, in ALS/FTD, there is a compromised stress granule response in disease-affected neurons due to impaired G3BP1 mRNA stability caused by TDP-43 nuclear depletion. These data implicate TDP-43 and G3BP1 loss of function as contributors to disease.

The prevalence of parasitic nematode infections are a major human and animal health concern. There are still no effective vaccines available, hence anthelmintic drugs have remained the cornerstone for prophylaxis and treatment. The repertoire of available anthelmintics is limited, with treatment relying heavily on three major chemical classes of anthelmintics. These are the imidazothiazoles/tetrahydropyrimidines (levamisole, pyrantel, morantel, oxantel); benzimidazoles (mebendazole, flubendazole, thiabendazole, albendazole); and the macrocyclic lactones (ivermectin, moxidectin, abamectin), all of which act on parasitic nematode ion channels, except for the benzimidazoles. Ion-channels are crucial components of excitable tissues and valuable targets for anthelmintics. Prolong treatment and incorrect use of these anthelmintics however, have led to the development of resistance worldwide. Additionally, the development of new anthelmintics is slow-paced, with only three drug classes being developed and approved for animal use in the since the year 2000. This includes the amino-acetonitrile (monepantel), cyclooctadepsipeptide (emodepside) and the spiroindole (derquantel). Hence, there is an urgent need for the development of new, more effective anthelmintic drugs that can alleviate the morbidity and mortality caused by existing parasite infections. Additionally, significant gaps in our understanding of anthelmintic resistance need to be improved so that we can provide practical solutions on improving drug efficacy and delaying the onset of resistance. We have confirmed the expression of four nicotinic acetylcholine receptor (nAChR) subunits: Asu-unc-38, Asu-unc-29, Asu-unc-63 and Asu-acr-8 that constitute the putative levamisole receptor in adult female Ascaris suum intestine. We then validated these findings by using RNAscope in situ hybridization to localize the subcellular distribution of the subunits in the intestine. Quantitative real-time PCR (qPCR) was also used to confirm the mRNA expression levels of each subunit in both muscle and intestine cells. To determine whether these subunits formed functional receptors that were responsive to cholinergic agonists, we employed calcium imaging. Our calcium imaging results demonstrated that both acetylcholine and levamisole elicited intracellular calcium responses in the intestinal tissue. These findings suggest that the presence of functional nAChRs in the intestine may not be limited to neuromuscular transmission, but an acetylcholine paracrine function. Hence, A. suum intestine can be a suitable target for therapeutic exploitation. Secondly, we expressed two receptor subtypes, namely Ode-levamisole and Ode pyrantel/tribendimidine from the pig parasite Oesophagostomum dentatum in Xenopus laevis oocytes. We demonstrated that compounds from the two macrocyclic lactone sub-family, the avermectins (abamectin and ivermectin) and the milbemycin, moxidectin are positive allosteric modulators (PAMs) on the Ode levamisole receptor. In contrast, abamectin and ivermectin acted as negative allosteric modulators (NAMs) on the Ode pyrantel/tribendimidine receptor subtype, while moxidectin maintained its PAM action. These findings suggest that the macrocyclic lactones are allosteric modulators of nAChRs and structural differences between each drug or the presence or absence of a subunit, namely ACR-8 may influence the allosteric modulatory effects. Hence combination therapy that includes macrocyclic lactones and cholinergic anthelmintics, might improve drug efficacy and delay anthelmintic resistance. Finally, we investigated the adaptation of Brugia malayi to levamisole exposure. We showed that B. malayi recovered motility with loss of sensitivity to levamisole within 4 hours of exposure. Molecular analysis also revealed up-regulation of mRNA levels for one AChR subunit, unc-38 and down-regulation of a gene that encodes for an ER retention protein, nra-2. Patch-clamp experiments on 4 hour recovered worms also showed that muscle responses to levamisole had desensitized. Knock down of nra-2 by RNAi resulted in faster recovery in motility, significant reduction in levamisole currents and no change in acetylcholine currents. This suggest that loss of NRA-2 facilitates the insertion of pentameric AChR subtypes in the muscle that are insensitive to levamisole, thus leading to faster recovery in motility in the presence of levamisole. Additionally, simultaneous knockdown of AChR subunits, namely, unc-38, acr-26 and acr-16, inhibited recovery of motility in the worms. These findings are notable and highlights the dynamic mechanisms used to by the parasite to vary AChR subunit composition that generates various receptor subtypes, thus facilitating recovery of motility and insensitivity to anthelmintic exposure (levamisole). This process of habituation can be interpreted as a mechanism of resistance that can be used by parasitic nematodes.

AXIN2 and LGR5 mark intestinal stem cells (ISCs) that require WNT/β-Catenin signaling for constant homeostatic proliferation. In contrast, AXIN2/LGR5+ pericentral hepatocytes show low proliferation rates despite a WNT/β-Catenin activity gradient required for metabolic liver zonation. The mechanisms restricting proliferation in AXIN2+ hepatocytes and metabolic gene expression in AXIN2+ ISCs remained elusive. We now show that restricted chromatin accessibility in ISCs prevents the expression of β-Catenin-regulated metabolic enzymes, whereas fine-tuning of WNT/β-Catenin activity by ZNRF3 and RNF43 restricts proliferation in chromatin-permissive AXIN2+ hepatocytes, while preserving metabolic function. ZNRF3 deletion promotes hepatocyte proliferation, which in turn becomes limited by RNF43 upregulation. Concomitant deletion of RNF43 in ZNRF3 mutant mice results in metabolic reprogramming of periportal hepatocytes and induces clonal expansion in a subset of hepatocytes, ultimately promoting liver tumors. Together, ZNRF3 and RNF43 cooperate to safeguard liver homeostasis by spatially and temporally restricting WNT/β-Catenin activity, balancing metabolic function and hepatocyte proliferation.

Methylmalonic acidemia (MMA) is a severe metabolic disorder most commonly caused by a mutation in the methylmalonyl-CoA mutase (MMUT) gene. Patients with MMA experience multisystemic disease manifestations and remain at risk for neurological disease progression, even after liver transplantation. Therefore, delivery of MMUT to the central nervous system (CNS) may provide patients with neuroprotection and, perhaps, therapeutic benefits. To specifically target the brain, we developed a neurotropic PHP.eB vector that used a CaMKII neuro-specific promoter to restrict the expression of the MMUT transgene in the neuraxis and delivered the adeno-associated virus (AAV) to mice with MMA. The PHP.eB vector transduced cells in multiple brain regions, including the striatum, and enabled high levels of expression of MMUT in the basal ganglia. Following the CNS-specific correction of MMUT expression, disease-related metabolites methylmalonic acid and 2-methylcitrate were significantly (p < 0.02) decreased in serum of treated MMA mice. Our results show that targeting MMUT expression to the CNS using a neurotropic capsid can decrease the circulating metabolite load in MMA and further highlight the benefit of extrahepatic correction for disorders of organic acid metabolism.

Bladder Cancer is the 9th most common malignancy in the world. Transitional cell carcinoma (TCC) is the most common of bladder cancers, occurring in 90% of cases. There has been no great model established to study TCC in vitro. In this study, we explore urine-derived, 3-dimensional, canine TCC organoids as a possible model to study TCC in vitro. After establishing the cell line, we subjected the 3-D cells to RNA in situ hybridization (RNAish) and cell viability assays. Overall, 3-D cell culture from urine samples of TCC diagnosed canines expressed RNA biomarkers in a similar manner to parent tumors via RNAish and showed more sensitivity to Cisplatin treatment when compared to 2-D human TCC cells. With further experimentation, canine TCC organoids could be an ideal model to study TCC in vitro.

Metabolic diseases are associated with an increased risk of severe COVID-19 and conversely, new-onset hyperglycemia and complications of preexisting diabetes have been observed in COVID-19 patients. Here, we performed a comprehensive analysis of pancreatic autopsy tissue from COVID-19 patients using immunofluorescence, immunohistochemistry, RNA scope and electron microscopy and detected SARS-CoV-2 viral infiltration of beta-cells in all patients. Using SARS-CoV-2 pseudoviruses, we confirmed that isolated human islet cells are permissive to infection. In eleven COVID-19 patients, we examined the expression of ACE2, TMPRSS and other receptors and factors, such as DPP4, HMBG1 and NRP1, that might facilitate virus entry. Whereas 70% of the COVID-19 patients expressed ACE2 in the vasculature, only 30% displayed ACE2-expression in beta-cells. Even in the absence of manifest new-onset diabetes, necroptotic cell death, immune cell infiltration and SARS-CoV-2 viral infection of pancreatic beta-cells may contribute to varying degrees of metabolic dysregulation in patients with COVID-19.

Porcine circovirus 3 (PCV3) was first discovered in 2016, almost concomitantly by two groups of researchers in the United States. The novel case was reported in a group of sows with chronic reproductive problems with clinical presentation alike porcine dermatitis and nephropathy syndrome (PDNS), where metagenomic sequencing revealed a genetically divergent porcine circovirus designated PCV3. The discovery of PCV3 in a PDNS case, which used to be considered as part of PCVAD attributed to PCV2 (porcine circovirus 2), has garnered attention and effort in further research of the novel virus. Just when an infectious molecular DNA clone of PCV3 has been developed and successfully used in an in vivo pathogenicity study, yet another novel PCV strain surfaced, designated PCV4 (porcine circovirus 4). So far, PCV3 has been reported in domestic swine population globally at low to moderate prevalence, from almost all sample types including organ tissues, faecal, semen and colostrum samples. PCV3 has been associated with a myriad of clinical presentations, from PDNS to porcine respiratory disease complex (PRDC). This review paper summarizes the studies on PCV3 to date, with focus on diagnosis.

xCT is the specific subunit of System xc-, an antiporter importing cystine while releasing glutamate. Although xCT expression has been found in the spinal cord, its expression and role after spinal cord injury (SCI) remain unknown. The aim of this study was to characterize the role of xCT on functional and histological outcomes following SCI induced in wild-type (xCT+/+) and in xCT-deficient mice (xCT-/-). In the normal mouse spinal cord, slc7a11/xCT mRNA was detected in meningeal fibroblasts, vascular mural cells, astrocytes, motor neurons and to a lesser extent in microglia. slc7a11/xCT gene and protein were upregulated within two weeks post-SCI. xCT-/- mice recovered muscular grip strength as well as pre-SCI weight faster than xCT+/+ mice. Histology of xCT-/- spinal cords revealed significantly more spared motor neurons and a higher number of quiescent microglia. In xCT-/- mice, inflammatory polarization shifted towards higher mRNA expression of ym1 and igf1 (anti-inflammatory) while lower levels of nox2 and tnf-a (pro-inflammatory). Although astrocyte polarization did not differ, we quantified an increased expression of lcn2 mRNA. Our results show that slc7a11/xCT is overexpressed early following SCI and is detrimental to motor neuron survival. xCT deletion modulates intraspinal glial activation by shifting towards an anti-inflammatory profile.