Sex differences in population dynamics during formation of kidney bacterial communities by uropathogenic Escherichia coli
McLellan, LK;Daugherty, AL;Hunstad, DA;
PMID: 33468577 | DOI: 10.1128/IAI.00716-20
Uropathogenic Escherichia coli (UPEC), the primary etiologic agent of urinary tract infections (UTIs), encounters a restrictive population bottleneck within the female mammalian bladder. Its genetic diversity is restricted during establishment of cystitis because successful UPEC must invade superficial bladder epithelial cells prior to forming clonal intracellular bacterial communities (IBCs). Here, we aimed to understand UPEC population dynamics during ascending pyelonephritis, namely formation of kidney bacterial communities (KBCs) in the renal tubular lumen and nucleation of renal abscesses. We inoculated the bladders of both male and female C3H/HeN mice, a background which features vesicoureteral reflux; we have previously shown that in this model, males develop severe, high-titer pyelonephritis and renal abscesses much more frequently than females. Mice were infected with 40 isogenic, PCR-tagged ("barcoded") UPEC strains, and tags remaining in bladder and kidneys were ascertained at intervals following infection. In contrast to females, males maintained a majority of strains within both the bladder and kidneys throughout the course of infection, indicating only a modest host-imposed bottleneck on overall population diversity during successful renal infection. Moreover, the diverse population in the infected male kidneys obscured any restrictive bottleneck in the male bladder. Finally, using RNA-in situ hybridization following mixed infections with isogenic UPEC bearing distinct markers, we found that despite their extracellular location (in the urinary space), KBCs are clonal in origin. This finding indicates that even with bulk reflux of infected bladder urine into the renal pelvis, successful ascension of UPEC to establish the tubular niche is an uncommon event.
An Atoh1 CRE knock-in mouse labels motor neurons involved in fine motor control
Ogujiofor, OW;Pop, IV;Espinosa, F;Durodoye, RO;Viacheslavov, ML;Jarvis, R;Landy, MA;Gurumurthy, CB;Lai, HC;
PMID: 33468540 | DOI: 10.1523/ENEURO.0221-20.2021
Motor neurons (MNs) innervating the digit muscles of the intrinsic hand and foot (IH and IF) control fine motor movements. The ability to reproducibly label specifically IH and IF MNs in mice would be a beneficial tool for studies focused on fine motor control. To this end, we find that a CRE knock-in mouse line of Atoh1, a developmentally expressed basic helix-loop-helix (bHLH) transcription factor, reliably expresses CRE-dependent reporter genes in approximately 60% of the IH and IF MNs. We determine that CRE-dependent expression in IH and IF MNs is ectopic because an Atoh1 mouse line driving FLPo recombinase does not label these MNs even though other Atoh1-lineage neurons in the intermediate spinal cord are reliably identified. Furthermore, the CRE-dependent reporter expression is enriched in the IH and IF MN pools with much sparser labeling of other limb-innervating MN pools such as the tibialis anterior, gastrocnemius, quadricep, and adductor. Lastly, we find that ectopic reporter expression begins postnatally and labels a mixture of alpha and gamma-MNs. Altogether, the Atoh1 CRE knock-in mouse strain might be a useful tool to explore the function and connectivity of MNs involved in fine motor control when combined with other genetic or viral strategies that can restrict labeling specifically to the IH and IF MNs. Accordingly, we provide an example of sparse labeling of IH and IF MNs using an intersectional genetic approach.Significance Statement Motor neurons (MNs) of the intrinsic hand and foot (IH and IF) are reproducibly labeled in an ectopic manner postnatally using a CRE knock-in mouse line of the basic helix-loop-helix (bHLH) transcription factor Atoh1, serving as a useful genetic tool for future studies of fine motor control.
SARS-CoV-2 leads to a small vessel endotheliitis in the heart
Maccio, U;Zinkernagel, AS;Shambat, SM;Zeng, X;Cathomas, G;Ruschitzka, F;Schuepbach, RA;Moch, H;Varga, Z;
PMID: 33422990 | DOI: 10.1016/j.ebiom.2020.103182
SARS-CoV-2 infection (COVID-19 disease) can induce systemic vascular involvement contributing to morbidity and mortality. SARS-CoV-2 targets epithelial and endothelial cells through the ACE2 receptor. The anatomical involvement of the coronary tree is not explored yet. Cardiac autopsy tissue of the entire coronary tree (main coronary arteries, epicardial arterioles/venules, epicardial capillaries) and epicardial nerves were analyzed in COVID-19 patients (n = 6). All anatomical regions were immunohistochemically tested for ACE2, TMPRSS2, CD147, CD45, CD3, CD4, CD8, CD68 and IL-6. COVID-19 negative patients with cardiovascular disease (n = 3) and influenza A (n = 6) served as controls. COVID-19 positive patients showed strong ACE2 / TMPRSS2 expression in capillaries and less in arterioles/venules. The main coronary arteries were virtually devoid of ACE2 receptor and had only mild intimal inflammation. Epicardial capillaries had a prominent lympho-monocytic endotheliitis, which was less pronounced in arterioles/venules. The lymphocytic-monocytic infiltrate strongly expressed CD4, CD45, CD68. Peri/epicardial nerves had strong ACE2 expression and lympho-monocytic inflammation. COVID-19 negative patients showed minimal vascular ACE2 expression and lacked endotheliitis or inflammatory reaction. ACE2 / TMPRSS2 expression and lymphomonocytic inflammation in COVID-19 disease increases crescentically towards the small vessels suggesting that COVID-19-induced endotheliitis is a small vessel vasculitis not involving the main coronaries. The inflammatory neuropathy of epicardial nerves in COVID-19 disease provides further evidence of an angio- and neurotrophic affinity of SARS-COV2 and might potentially contribute to the understanding of the high prevalence of cardiac complications such as myocardial injury and arrhythmias in COVID-19. No external funding was necessary for this study.
Proceedings of the National Academy of Sciences of the United States of America
Nambulli, S;Rennick, LJ;Acciardo, AS;Tilston-Lunel, NL;Ho, G;Crossland, NA;Hardcastle, K;Nieto, B;Bainbridge, G;Williams, T;Sharp, CR;Duprex, WP;
PMID: 36251995 | DOI: 10.1073/pnas.2209405119
Feline morbillivirus (FeMV) is a recently discovered pathogen of domestic cats and has been classified as a morbillivirus in the Paramyxovirus family. We determined the complete sequence of FeMVUS5 directly from an FeMV-positive urine sample without virus isolation or cell passage. Sequence analysis of the viral genome revealed potential divergence from characteristics of archetypal morbilliviruses. First, the virus lacks the canonical polybasic furin cleavage signal in the fusion (F) glycoprotein. Second, conserved amino acids in the hemagglutinin (H) glycoprotein used by all other morbilliviruses for binding and/or fusion activation with the cellular receptor CD150 (signaling lymphocyte activation molecule [SLAM]/F1) are absent. We show that, despite this sequence divergence, FeMV H glycoprotein uses feline CD150 as a receptor and cannot use human CD150. We demonstrate that the protease responsible for cleaving the FeMV F glycoprotein is a cathepsin, making FeMV a unique morbillivirus and more similar to the closely related zoonotic Nipah and Hendra viruses. We developed a reverse genetics system for FeMVUS5 and generated recombinant viruses expressing Venus fluorescent protein from an additional transcription unit located either between the phospho-protein (P) and matrix (M) genes or the H and large (L) genes of the genome. We used these recombinant FeMVs to establish a natural infection and demonstrate that FeMV causes an acute morbillivirus-like disease in the cat. Virus was shed in the urine and detectable in the kidneys at later time points. This opens the door for long-term studies to address the postulated role of this morbillivirus in the development of chronic kidney disease.
European neuropsychopharmacology : the journal of the European College of Neuropsychopharmacology
Begni, V;Pisano, I;Marizzoni, M;Marchisella, F;Creutzberg, KC;De Rosa, F;Cattaneo, A;Gruca, P;Litwa, E;Papp, M;Riva, MA;
PMID: 35830759 | DOI: 10.1016/j.euroneuro.2022.06.005
Chronic stress represents a major contributor for the development of mental illness. This study aimed to investigate how animals exposed to chronic mild stress (CMS) responded to an acute stress (AS), as a vulnerability's challenge, and to establish the potential effects of the antipsychotic drug lurasidone on such mechanisms. Adult male Wistar rats were exposed or not (controls) to a CMS paradigm for 7 weeks. Starting from the end of week 2, animals were randomized to receive vehicle or lurasidone for 5 weeks. Sucrose intake was used to measure anhedonia. At the end, half of the animals were exposed to an acute stress before sacrifice. Exposure to CMS produced a significant reduction in sucrose consumption, whereas lurasidone progressively normalized such alteration. We found that exposure to AS produced an upregulation of Brain derived neurotrophic factor (Bdnf) in the prefrontal cortex of controls animals. This response was impaired in CMS rats and restored by lurasidone treatment. While in control animals, AS-induced increase of Bdnf mRNA levels was specific for Parvalbumin cells, CMS rats treated with lurasidone show a significant upregulation of Bdnf in pyramidal cells. Furthermore, when investigating the activation of different brain regions, CMS rats showed an impairment in the global response to the acute stressor, that was largely restored by lurasidone treatment. Our results suggest that lurasidone treatment in CMS rats may regulate specific circuits and mechanisms, which will ultimately contribute to boost resilience under stressful challenges.
Eom, K;Lee, HR;Hyun, JH;An, H;Lee, YS;Ho, WK;Lee, SH;
PMID: 34964210 | DOI: 10.1002/hipo.23400
The associative network of hippocampal CA3 is thought to contribute to rapid formation of contextual memory from one-trial learning, but the network mechanisms underlying decorrelation of neuronal ensembles in CA3 is largely unknown. Kv1.2 expressions in rodent CA3 pyramidal cells (CA3-PCs) are polarized to distal apical dendrites, and its downregulation specifically enhances dendritic responses to perforant pathway (PP) synaptic inputs. We found that haploinsufficiency of Kv1.2 (Kcna2+/-) in CA3-PCs, but not Kv1.1 (Kcna1+/-), lowers the threshold for long-term potentiation (LTP) at PP-CA3 synapses, and that the Kcna2+/- mice are normal in discrimination of distinct contexts but impaired in discrimination of similar but slightly distinct contexts. We further examined the neuronal ensembles in CA3 and dentate gyrus (DG), which represent the two similar contexts using in situ hybridization of immediate early genes, Homer1a and Arc. The size and overlap of CA3 ensembles activated by the first visit to the similar contexts were not different between wild type and Kcna2+/- mice, but these ensemble parameters diverged over training days between genotypes, suggesting that abnormal plastic changes at PP-CA3 synapses of Kcna2+/- mice is responsible for the impaired pattern separation. Unlike CA3, DG ensembles were not different between two genotype mice. The DG ensembles were already separated on the first day, and their overlap did not further evolve. Eventually, the Kcna2+/- mice exhibited larger CA3 ensemble size and overlap upon retrieval of two contexts, compared to wild type or Kcna1+/- mice. These results suggest that sparse LTP at PP-CA3 synapse probably supervised by mossy fiber inputs is essential for gradual decorrelation of CA3 ensembles.
European archives of oto-rhino-laryngology : official journal of the European Federation of Oto-Rhino-Laryngological Societies (EUFOS) : affiliated with the German Society for Oto-Rhino-Laryngology - Head and Neck Surgery
Jiromaru, R;Yasumatsu, R;Yamamoto, H;Kuga, R;Hongo, T;Nakano, T;Manako, T;Hashimoto, K;Wakasaki, T;Matsuo, M;Nakagawa, T;
PMID: 34978590 | DOI: 10.1007/s00405-021-07236-z
We herein report the treatment outcome of oropharyngeal squamous cell carcinoma (OPSCC) at Kyushu University Hospital, the total number of OPSCC cases, and changes in the proportion of human papilloma virus (HPV)-related carcinomas over time.We performed a retrospective analysis of 237 cases treated for OPSCC at Kyushu University Hospital between 2013 and 2019. We performed HPV-mRNA in situ hybridization and p16 immunohistochemistry.This study included 197 males (82.1%) and 40 females (17.9%). The disease-specific, progression-free and overall survival (OS) were 69%, 62% and 61%, respectively, over the decade-long study period. p16-Immunohistochemistory and highrisk HPV mRNA in situ hybridization were positive in 114 (48.1%) and 105 (44.3%) cases, respectively. The number of HPV-related OPSCC cases increased according to an annual analysis. HPV+ cases had a significantly better prognosis than HPV- cases. In addition, p16+/HPV- cases had a significantly worse prognosis than p16+/HPV+ cases (OS: p = 0.0484). HPV+ OPSCC cases were associated with a younger age (< 60 years old) (p = 0.0429), non-smoker (p = 0.0001), lateral tumor site (< 0.00001), lymphoid metastasis (< 0.0001) and low clinical stage (< 0.0001).The frequency of HPV-related OPSCC cases is increasing in Japan as well as worldwide, and such cases are characterized by no smoking habit, a young age, and a good prognosis. Even in p16+ OPSCC, HPV- cases had a poor prognosis, suggesting the importance of accurate HPV determination. To determine the intensity of treatment for HPV-related and non-related OPSCC, it is necessary to accumulate cases for the accurate HPV determination and comparison of treatment effects.
Banu, M;Dovas, A;Argenziano, M;Zhao, W;Higgins, D;Upadhyayula, P;Mahajan, A;Humala, N;Nguyen, T;Zandkarimi, F;Siegelin, M;Brent, S;Sims, P;Bruce, J;Canoll, P;
| DOI: 10.1093/neuonc/noab196.852
Diversity is a key feature in the glioma ecosystem. Adaptation to a changing tumor microenvironment is achieved through cellular and metabolic plasticity. Here we show that slow-cycling, astrocyte-like glioma cell subpopulations activate distinct metabolic programs, rendering them susceptible to novel treatments. We performed multi-omics analysis on transgenic murine glioma models to characterize cellular heterogeneity. Bulk RNAseq on targeted time-dependent biopsies combined with scRNAseq uncovered distinct tumor cell populations, including a quiescent, astrocyte-like population relatively insensitive to conventional chemotherapy targeting proliferating cells. Using scRNAseq, we identified a persistently conserved astrocytic population in human IDH1-mt/wt high-grade gliomas. This astrocytic tumor population was more abundant in mouse models with constitutive Notch activation, however it was associated with alterations in several other transcriptional programs, suggesting that targeted therapies would likely be ineffective at eradicating it. Gene ontology analysis revealed enrichment in mitochondrial genes specifically regulating oxidative phosphorylation and tricarboxylic acid cycle. Energetic, lipidomic and metabolomic analyses revealed significant mitochondrial β-fatty acid oxidation and lipid catabolism, with less effective oxygen consumption rate and higher basal oxidative stress. Furthermore, this astrocytic tumor population had depleted levels of basal GSH and was more sensitive to reactive oxygen species. Leveraging this metabolic vulnerability, we performed drug screens and found that therapeutic inhibition of complex I or GPX4 was highly effective and synergistic. GPX4 inhibition induced ferroptosis, a newly-discovered form of programmed non-necroptotic cell death mediated by iron-driven lipid peroxidation. Using scRNAseq and RNAscope on ex vivo slice cultures from murine and human gliomas, we found that GPX4 inhibition and ferroptosis induction in the glioma microenvironment selectively eradicated the quiescent astrocytic subpopulation whereas proliferating glioma were less sensitive. Our data therefore supports a novel treatment paradigm, employing metabolic strategies, such as ferroptosis, in conjunction with chemotherapy and RT to target distinct tumor cell populations with different therapeutic vulnerabilities.
Journal of Cystic Fibrosis
Bedwell, D;Sharma, J;Du, M;Wong, E;Mutyam, V;Li, Y;Chen, J;Wangen, J;Thrasher, K;Fu, L;Peng, N;Tang, L;Liu, K;Mathew, B;Bostwick, B;Augelli-Szafran, C;Bihler, H;Liang, F;Mahiou, J;Saltz, J;Rab, A;Hong, J;Sorscher, E;Mendenhall, E;Coppola, C;Keeling, K;Green, R;Mense, M;Suto, M;Rowe, S;
| DOI: 10.1016/S1569-1993(21)01955-X
Background: Although recently developed CFTR modulator drugs can alleviate cystic fibrosis (CF) in the majority of patients, CF patients with mutations that form premature termination codons (PTCs) cannot benefit from modulator therapies. PTCs terminate translation before a full-length CF transmembrane regulator (CFTR) protein can be generated. In addition, a PTC can trigger nonsense-mediated mRNA decay of the CFTR transcript, further reducing expression of CFTR protein. Together, these 2 PTCmediated events result in negligible CFTR protein, abrogating the usefulness of CFTR modulators, whose action requires CFTR expression. Nonsense suppression therapy uses small molecules to suppress translation termination at in-frame PTCs (nonsense mutations) to restore partial levels of full-length, functional CFTR protein. Methods: Although some compounds have been identified that can suppress translation termination at PTCs (also called readthrough), poor efficacy of current readthrough agents prompted us to search for more effective compounds. To this end, we developed a NanoLuc reporter system in Fischer rat thyroid cells that responds to readthrough and nonsensemediated mRNA decay. This assay was used to screen 771 345 lowmolecular-weight compounds. Results: Of the 180 compounds identified with readthrough activity, SRI37240 and its more potent derivative SRI-41315 suppressed multiple CFassociated PTCs in immortalized and primary human bronchial epithelial cells, restoring partial CFTR expression and function. Mechanistically, we found that these compounds induce a prolonged pause at stop codons and suppress PTCs by targeting the termination factor eRF1 to the proteasome and thus reducing its abundance. Moreover, SRI-41315 enhances aminoglycoside-mediated readthrough, leading to synergistic increases in CFTR activity. Conclusion: SRI-37240 and SRI-41315 are the first pharmacological agents known to alter eRF1 levels and thus represent a new class of readthrough compounds that could potentially be used as part of a nonsense suppression therapy to treat genetic diseases in patients who carry PTCs
RNA-sequencing and immunofluorescence of the myotendinous junction of mature horses and humans
American journal of physiology. Cell physiology
Jakobsen, JR;Schjerling, P;Svensson, RB;Buhl, R;Carstensen, H;Koch, M;Rindom Krogsgaard, MR;Kjaer, M;Mackey, AL;
PMID: 34260300 | DOI: 10.1152/ajpcell.00218.2021
The myotendinous junction (MTJ) is a specialised interface for transmitting high forces between muscle and tendon and yet the MTJ is a common site of strain injury with a high recurrence rate. The aim of this study was to identify previously unknown MTJ components in mature animals and humans. Samples were obtained from the superficial digital flexor (SDF) muscle-tendon interface of 20 horses and the tissue was separated through a sequential cryo-sectioning approach into muscle, MTJ (muscle tissue enriched in myofiber tips attached to the tendon), and tendon fractions. RT-PCR was performed for genes known to be expressed in the three tissue fractions and t-SNE plots were used to select the muscle, MTJ and tendon samples from 5 horses for RNA-sequencing. The expression of previously known and unknown genes identified through RNA-sequencing was studied by immunofluorescence on human hamstring MTJ tissue. The main finding was that RNA-sequencing identified expression of a panel of 61 genes enriched at the MTJ. 48 of these genes were novel for the MTJ, and 13 genes had been reported to be associated with the MTJ in earlier studies. The expression of known (COL22A1, NCAM, POSTN, NES, OSTN) and previously undescribed (MNS1 and LCT) MTJ genes was confirmed at the protein level by immunofluorescence on tissue sections of human MTJ. In conclusion, in muscle-tendon interface tissue enriched with myofiber tips, we identified expression of previously unknown MTJ genes representing diverse biological processes, which may be important in the maintenance of the specialized MTJ.
Porcine Epidemic Diarrhea Virus Induces Vero Cell Apoptosis via the p53-PUMA Signaling Pathway
Yang, L;Wang, C;Shu, J;Feng, H;He, Y;Chen, J;Shu, J;
PMID: 34202551 | DOI: 10.3390/v13071218
Porcine Epidemic Diarrhea Virus (PEDV) is the causative agent of swine epidemic diarrhea. In order to study the pathogenic mechanism of PEDV, PEDV was inoculated into Vero cells cultured in vitro, and the total RNA of Vero cells was extracted to construct a library for Illumina high-throughput sequencing and screening of differentially expressed genes (p < 0.05). Five differentially expressed genes for qRT-PCR verification analysis were randomly selected, and the verification results were consistent with the transcriptome sequencing results. The Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway enrichment analysis was performed on the differentially expressed genes screened above. The results showed that the target gene annotations of differentially expressed genes in the African green monkey genome were mainly enriched in the TNF signaling pathway, the P53 signaling pathway, the Jak-STAT signaling pathway, the MAPK signaling pathway, and immune inflammation. In addition, it has been reported that Puma can promote apoptosis and is a key mediator of P53-dependent and non-dependent apoptosis pathways. However, there is no report that PEDV infection can activate Puma and induce apoptosis in a P53-dependent pathway. It was found by flow cytometry that PEDV infection induced apoptosis, and by Western Blotting detection, PEDV infection significantly increased the expression of p53, BAX, and Puma apoptosis-related proteins. Treatment Vero cells with the p53 inhibitor, PFT-α, could significantly inhibit PEDV-induced apoptosis. Studies have shown that PEDV infection can activate Puma and induce apoptosis in a P53-dependent pathway. These findings provide data support for further elucidating the pathogenic mechanism of PEDV and developing an effective vaccine against PEDV.
Verrucous Carcinoma of the Esophagus Is A Genetically Distinct Subtype of Esophageal Squamous Cell Carcinoma
Isidro, RA;Dong, F;Hornick, JL;Wee, JO;Agoston, A;Patil, DT;Deshpande, V;Zhao, L;
PMID: 33960520 | DOI: 10.1111/his.14395
Esophageal verrucous carcinoma (VSCC) is a rare and morphologically distinct type of esophageal squamous cell carcinoma (SCC). Diagnosing VSCC on biopsy material is challenging given the lack of significant atypia and the presence of keratinizing epithelium and exophytic growth. The molecular pathogenesis of VSCC remains unclear. The aim of this study was to characterize the genomic landscape of VSCC in comparison to conventional esophageal SCC. Three cases of VSCC from the Brigham and Women's Hospital pathology archive were identified. Formalin-fixed, paraffin-embedded (FFPE) tumor tissue was used for p16 immunohistochemistry (IHC), high-risk HPV in situ mRNA hybridization (ISH), and DNA isolation. Tumor DNA was sequenced using a targeted massively parallel sequencing assay enriched for cancer-associated genes. Three additional cases of VSCC were identified by image review of The Cancer Genome Atlas (TCGA) esophageal SCC cohort. VSCC cases were negative for p16 IHC and high-risk HPV ISH. TP53 mutations (p<0.001) and copy number variants (CNVs) for CDKN2A (p<0.001), CDKN2B (p<0.01) and CCND1 (p<0.01) were absent in VSCC and significantly less frequent in comparison to conventional SCC. Five VSCC cases featured SMARCA4 missense mutations or inframe deletions compared to only 4/88 conventional SCC cases (p<0.001). VSCC featured driver mutations in PIK3CA, HRAS, and GNAS. Recurrent CNVs were rare in VSCC. VSCC is not only morphologically but also genetically distinct from conventional esophageal SCC, featuring frequent SMARCA4 mutations and infrequent TP53 mutations or CDKN2A/B CNVs. Molecular findings may aid in establishing the challenging diagnosis of VSCC. This article is protected by