Amyloplast is involved in the MIZ1-modulated root hydrotropism.

Roots exhibit hydrotropism in response to moisture gradients, with the hydrotropism-related gene Mizu-kussei1 (MIZ1) playing a role in regulating root hydrotropism in an oblique orientation. However, the mechanisms underlying MIZ1-regulated root hydrotropism are not well understood. In this study, we employed obliquely oriented experimental systems to investigate root hydrotropism in Arabidopsis. We found that the miz1 mutant displays reduced root hydrotropism but increased root gravitropism following hydrostimulation, as compared to wild-type plants. Conversely, overexpression of AtMIZ1 leads to enhanced root hydrotropism but decreased root gravitropism following hydrostimulation, as compared to wild-type plants. Using co-immunoprecipitation followed by mass spectrometry (IP-MS), we explored proteins that interact with AtMIZ1, and we identified PGMC1 co-immunoprecipitated with MIZ1 in vivo. Furthermore, the miz1 mutant exhibited higher expression of the PGMC1 gene and increased phosphoglucomutase (PGM) activity, while AtMIZ1 overexpressors resulted in lower expression of the PGMC1 gene, reduced amyloplast amount, and reduced PGM activity in comparison to wild-type roots. In addition, different Arabidopsis natural accessions having difference in their hydrotropic response demonstrated expression level of PGMC1 was negatively correlated with hydrotropic root curvature and AtMIZ1 expression. Our results provide valuable insights into the role of amyloplast in MIZ1-regulated root hydrotropism.

Brain tropism acquisition: The spatial dynamics and evolution of a measles virus collective infectious unit that drove lethal subacute sclerosing panencephalitis.

It is increasingly appreciated that pathogens can spread as infectious units constituted by multiple, genetically diverse genomes, also called collective infectious units or genome collectives. However, genetic characterization of the spatial dynamics of collective infectious units in animal hosts is demanding, and it is rarely feasible in humans. Measles virus (MeV), whose spread in lymphatic tissues and airway epithelia relies on collective infectious units, can, in rare cases, cause subacute sclerosing panencephalitis (SSPE), a lethal human brain disease. In different SSPE cases, MeV acquisition of brain tropism has been attributed to mutations affecting either the fusion or the matrix protein, or both, but the overarching mechanism driving brain adaptation is not understood. Here we analyzed MeV RNA from several spatially distinct brain regions of an individual who succumbed to SSPE. Surprisingly, we identified two major MeV genome subpopulations present at variable frequencies in all 15 brain specimens examined. Both genome types accumulated mutations like those shown to favor receptor-independent cell-cell spread in other SSPE cases. Most infected cells carried both genome types, suggesting the possibility of genetic complementation. We cannot definitively chart the history of the spread of this virus in the brain, but several observations suggest that mutant genomes generated in the frontal cortex moved outwards as a collective and diversified. During diversification, mutations affecting the cytoplasmic tails of both viral envelope proteins emerged and fluctuated in frequency across genetic backgrounds, suggesting convergent and potentially frequency-dependent evolution for modulation of fusogenicity. We propose that a collective infectious unit drove MeV pathogenesis in this brain. Re-examination of published data suggests that similar processes may have occurred in other SSPE cases. Our studies provide a primer for analyses of the evolution of collective infectious units of other pathogens that cause lethal disease in humans.

Auxin biosynthesis, transport, and response directly attenuate hydrotropism in the latter stages to fine-tune root growth direction in Arabidopsis.

Roots detect water potential gradients in the soil and orient toward moister areas, a response known as hydrotropism that aids drought avoidance. Although auxin is crucial in tropism, its polar transport is not essential for hydrotropism in Arabidopsis. Moreover, antiauxin treatments in Arabidopsis produced inconsistent outcomes: some studies indicated auxin action was necessary while others did not. In this study, we examined auxin's physiological role in hydrotropism. We found that inhibiting auxin biosynthesis or transport intensified hydrotropic bending not only in wild-type, but also in hydrotropism defective mutants, namely miz1-1 and miz2 plants. Given that miz1-1 and miz2 exhibited compromised hydrotropism even under clinorotated conditions, we infer that auxin biosynthesis and transport directly suppress hydrotropism. Additionally, tir1-10, afb1-3, and afb2-3 displayed augmented hydrotropism. We observed a significant delay in hydrotropic bending in arf7-1arf19-1, suggesting that ARF7 and ARF19 amplify hydrotropism in its early stages. To discern the functional ties of ARF7/19 with MIZ1 and MIZ2, we studied the hydrotropic phenotypes of arf7-1arf19-1miz1-1 and arf7-1arf19-1miz2. Both triple mutants had diminished early-stage hydrotropism yet showed partial but significant recovery in the later stages. Given MIZ1's role in reducing auxin levels and MIZ2's essentiality for MIZ1 functionality, we conclude that auxin inhibits hydrotropism downstream of MIZ1 in later stages to refine root bending. Furthermore, it is posited that gene expression driven by ARF7 and ARF19 is pivotal for early-stage root hydrotropism.

MIZ1 acts downstream of PGM1 in regulating root hydrotropism.

The MIZ1 play an important role in root hydrotropism. However, the relationship between MIZ1-regulated hydrotropism and amyloplast-mediated gravitropism remain largely unclear. Here, we generated the miz1/pgm1 double mutants by crossing the non-hydrotropic miz1 mutant with the amyloplast-defective pgm1 mutant, which lacks gravitropic response. Our results showed that the miz1/pgm1 mutants exhibited a significant reduction in amyloplast and gravitropic bending, while maintaining a similar ahydrotropic phenotype as the miz1 single mutant. These findings suggest that MIZ1 plays a role in hydrotropism downstream of PGM1. Understanding the mechanisms of interaction between hydrotropism and gravitropism is crucial for comprehending the rooting patterns of plants in natural conditions. The counteracting relationship between root hydrotropism and gravitropism in the miz1 mutant should receive attention in this field, particularly considering the interference from gravitropism on Earth.

Functional gene delivery to and across brain vasculature of systemic AAVs with endothelial-specific tropism in rodents and broad tropism in primates.

Delivering genes to and across the brain vasculature efficiently and specifically across species remains a critical challenge for addressing neurological diseases. We have evolved adeno-associated virus (AAV9) capsids into vectors that transduce brain endothelial cells specifically and efficiently following systemic administration in wild-type mice with diverse genetic backgrounds, and in rats. These AAVs also exhibit superior transduction of the CNS across non-human primates (marmosets and rhesus macaques), and in ex vivo human brain slices, although the endothelial tropism is not conserved across species. The capsid modifications translate from AAV9 to other serotypes such as AAV1 and AAV-DJ, enabling serotype switching for sequential AAV administration in mice. We demonstrate that the endothelial-specific mouse capsids can be used to genetically engineer the blood-brain barrier by transforming the mouse brain vasculature into a functional biofactory. We apply this approach to Hevin knockout mice, where AAV-X1-mediated ectopic expression of the synaptogenic protein Sparcl1/Hevin in brain endothelial cells rescued synaptic deficits.

Characterization of a Bioengineered AAV3B Capsid Variant with Enhanced Hepatocyte Tropism and Immune Evasion.

Capsid engineering of adeno-associated virus (AAV) can surmount current limitations to gene therapy such as broad tissue tropism, low transduction efficiency, or pre-existing neutralizing antibodies (NAb) that restrict patient eligibility. We previously generated an AAV3B combinatorial capsid library by integrating rational design and directed evolution with the aim of improving hepatotropism. A potential isolate, AAV3B-DE5, gained a selective proliferative advantage over five rounds of iterative selection in hepatocyte spheroid cultures. In this study, we reanalyzed our original dataset derived from the AAV3B combinatorial library and isolated variants from earlier (one to three) rounds of selection, with the assumption that variants with faster replication kinetics are not necessarily the most efficient transducers. We identified a potential candidate, AAV3B-V04, which demonstrated significantly enhanced transduction in mouse-passaged primary human hepatocytes as well as in humanized liver chimeric mice, compared to the parental AAV3B or the previously described isolate, AAV3B-DE5. Interestingly, the AAV3B-V04 capsid variant exhibited significantly reduced seroreactivity to pooled or individual human serum samples. Forty-four percent of serum samples with pre-existing NAbs to AAV3B had 5- to 20-fold lower reciprocal NAb titers to AAV3B-V04. AAV3B-V04 has only nine amino acid substitutions, clustered in variable region IV compared to AAV3B, indicating the importance of the loops at the top of the three-fold protrusions in determining both transduction efficiency and immunogenicity. This study highlights the effectiveness of rational design combined with targeted selection for enhanced AAV transduction via molecular evolution approaches. Our findings support the concept of limiting selection rounds to isolate the best transducing AAV3B variant without outgrowth of faster replicating candidates. We conclude that AAV3B-V04 provides advantages such as improved human hepatocyte tropism and immune evasion and propose its utility as a superior candidate for liver gene therapy.

Spatial transcriptomics for profiling the tropism of viral vectors in tissues.

A barrier to advancing engineered adeno-associated viral vectors (AAVs) for precision access to cell subtypes is a lack of high-throughput, high-resolution assays to characterize in vivo transduction profiles. In this study, we developed an ultrasensitive, sequential fluorescence in situ hybridization (USeqFISH) method for spatial transcriptomic profiling of endogenous and viral RNA with a short barcode in intact tissue volumes by integrating hydrogel-based tissue clearing, enhanced signal amplification and multiplexing using sequential labeling. Using USeqFISH, we investigated the transduction and cell subtype tropisms across mouse brain regions of six systemic AAVs, including AAV-PHP.AX, a new variant that transduces robustly and efficiently across neurons and astrocytes. Here we reveal distinct cell subtype biases of each AAV variant, including a bias of AAV-PHP.N toward excitatory neurons. USeqFISH also enables profiling of pooled regulatory cargos, as we show for a 13-variant pool of microRNA target sites in AAV genomes. Lastly, we demonstrate potential applications of USeqFISH for in situ AAV profiling and multimodal single-cell analysis in non-human primates.

Diversity of root hydrotropism among natural variants of Arabidopsis thaliana.

Root gravitropism affects root hydrotropism. The interference intensity of root gravitropism with root hydrotropism differs among plant species. However, these differences have not been well compared within a single plant species. In this study, we compared root hydrotropism in various natural variants of Arabidopsis under stationary conditions. As a result, we detected a range of root hydrotropism under stationary conditions among natural Arabidopsis variants. Comparison of root gravitropism and root hydrotropism among several Arabidopsis natural variants classified natural variants that decreased root hydrotropism into two types; namely one type that expresses root gravitropism and root hydrotropism weaker than Col-0, and the other type that expresses weaker root hydrotropism than Col-0 but expresses similar root gravitropism with Col-0. However, root hydrotropism of all examined Arabidopsis natural variants was facilitated by clinorotation. These results suggested that the interference of root gravitropism with root hydrotropism is conserved among Arabidopsis natural variants, although the intensity of root gravitropism interference with root hydrotropism differs.

Gene Expression Profile in Primary Tumor Is Associated with Brain-Tropism of Metastasis from Lung Adenocarcinoma.

Lung adenocarcinoma has a strong propensity to metastasize to the brain. The brain metastases are difficult to treat and can cause significant morbidity and mortality. Identifying patients with increased risk of developing brain metastasis can assist medical decision-making, facilitating a closer surveillance or justifying a preventive treatment. We analyzed 27 lung adenocarcinoma patients who received a primary lung tumor resection and developed metastases within 5 years after the surgery. Among these patients, 16 developed brain metastases and 11 developed non-brain metastases only. We performed targeted DNA sequencing, RNA sequencing and immunohistochemistry to characterize the difference between the primary tumors. We also compared our findings to the published data of brain-tropic and non-brain-tropic lung adenocarcinoma cell lines. The results demonstrated that the targeted tumor DNA sequencing did not reveal a significant difference between the groups, but the RNA sequencing identified 390 differentially expressed genes. A gene expression signature including CDKN2A could identify 100% of brain-metastasizing tumors with a 91% specificity. However, when compared to the differentially expressed genes between brain-tropic and non-brain-tropic lung cancer cell lines, a different set of genes was shared between the patient data and the cell line data, which include many genes implicated in the cancer-glia/neuron interaction. Our findings indicate that it is possible to identify lung adenocarcinoma patients at the highest risk for brain metastasis by analyzing the primary tumor. Further investigation is required to elucidate the mechanism behind these associations and to identify potential treatment targets.

Ten emerging SARS-CoV-2 spike variants exhibit variable infectivity, animal tropism, and antibody neutralization.

Emerging mutations in SARS-CoV-2 cause several waves of COVID-19 pandemic. Here we investigate the infectivity and antigenicity of ten emerging SARS-CoV-2 variants-B.1.1.298, B.1.1.7(Alpha), B.1.351(Beta), P.1(Gamma), P.2(Zeta), B.1.429(Epsilon), B.1.525(Eta), B.1.526-1(Iota), B.1.526-2(Iota), B.1.1.318-and seven corresponding single amino acid mutations in the receptor-binding domain using SARS-CoV-2 pseudovirus. The results indicate that the pseudovirus of most of the SARS-CoV-2 variants (except B.1.1.298) display slightly increased infectivity in human and monkey cell lines, especially B.1.351, B.1.525 and B.1.526 in Calu-3 cells. The K417N/T, N501Y, or E484K-carrying variants exhibit significantly increased abilities to infect mouse ACE2-overexpressing cells. The activities of furin, TMPRSS2, and cathepsin L are increased against most of the variants. RBD amino acid mutations comprising K417T/N, L452R, Y453F, S477N, E484K, and N501Y cause significant immune escape from 11 of 13 monoclonal antibodies. However, the resistance to neutralization by convalescent serum or vaccines elicited serum is mainly caused by the E484K mutation. The convalescent serum from B.1.1.7- and B.1.351-infected patients neutralized the variants themselves better than other SARS-CoV-2 variants. Our study provides insights regarding therapeutic antibodies and vaccines, and highlights the importance of E484K mutation.

Extended in vivo transcriptomes of two ascoviruses with different tissue tropisms reveal alternative mechanisms for enhancing virus reproduction in hemolymph.

Ascoviruses are large dsDNA viruses characterized by the extraordinary changes they induce in cellular pathogenesis and architecture whereby after nuclear lysis and extensive hypertrophy, each cell is cleaved into numerous vesicles for virion reproduction. However, the level of viral replication and transcription in vesicles compared to other host tissues remains uncertain. Therefore, we applied RNA-Sequencing to compare the temporal transcriptome of Spodoptera frugiperda ascovirus (SfAV) and Trichoplusia ni ascovirus (TnAV) at 7, 14, and 21 days post-infection (dpi). We found most transcription occurred in viral vesicles, not in initial tissues infected, a remarkably novel reproduction mechanism compared to all other viruses and most other intracellular pathogens. Specifically, the highest level of viral gene expression occurred in hemolymph, for TnAV at 7 dpi, and SfAV at 14 dpi. Moreover, we found that host immune genes were partially down-regulated in hemolymph, where most viral replication occurred in highly dense accumulations of vesicles.

Identification of a special cell type as a determinant of the kidney tropism of SARS-CoV-2.

The kidney tropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been well-validated clinically and often leads to various forms of renal damage in coronavirus disease-2019 (COVID-19) patients. However, the underlying mechanisms and diagnostic approaches remain to be determined. We interrogated the expression of virus-related host factors in single-cell RNA sequencing (scRNA-seq) datasets of normal human kidneys and kidneys with pre-existing diseases and validated the results with urinary proteomics of COVID-19 patients and healthy individuals. We also assessed the effects of genetic variants on kidney susceptibility using expression quantitative trait loci (eQTLs) databases. We identified a subtype of tubular cells, which we named PT-3 cells, as being vulnerable to SARS-CoV-2 infections in the kidneys. PT-3 cells were enriched in viral entry factors and replication and assembly machinery but lacked antiviral restriction factors. Immunohistochemistry confirmed positive staining of PT-3 cell marker SCL36A2 on kidney sections from COVID-19 patients. Urinary proteomic analyses of COVID-19 patients revealed that markers of PT-3 cells were significantly increased, along with elevated viral receptor angiotensin-converting enzyme 2. We further found that the proportion of PT-3 cells increased in diabetic nephropathy but decreased in kidney allografts and lupus nephropathy, suggesting that kidney susceptibility varied among these diseases. We finally identified several eQTLs that regulate the expression of host factors in kidney cells. PT-3 cells may represent a key determinant for the kidney tropism of SARS-CoV-2, and detection of PT-3 cells may be used to assess the risk of renal infection during COVID-19.

Translational adaptation of human viruses to the tissues they infect.

Viruses need to hijack the translational machinery of the host cell for a productive infection to happen. However, given the dynamic landscape of tRNA pools among tissues, it is unclear whether different viruses infecting different tissues have adapted their codon usage toward their tropism. Here, we collect the coding sequences of 502 human-infecting viruses and determine that tropism explains changes in codon usage. Using the tRNA abundances across 23 human tissues from The Cancer Genome Atlas (TCGA), we build an in silico model of translational efficiency that validates the correspondence of the viral codon usage with the translational machinery of their tropism. For instance, we detect that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is specifically adapted to the upper respiratory tract and alveoli. Furthermore, this correspondence is specifically defined in early viral proteins. The observed tissue-specific translational efficiency could be useful for the development of antiviral therapies and vaccines.

Evidence for root adaptation to a spatially discontinuous water availability in the absence of external water potential gradients.

We hereby show that root systems adapt to a spatially discontinuous pattern of water availability even when the gradients of water potential across them are vanishingly small. A paper microfluidic approach allowed us to expose the entire root system of Brassica rapa plants to a square array of water sources, separated by dry areas. Gradients in the concentration of water vapor across the root system were as small as 10-4⋅mM⋅m-1 (∼4 orders of magnitude smaller than in conventional hydrotropism assays). Despite such minuscule gradients (which greatly limit the possible influence of the well-understood gradient-driven hydrotropic response), our results show that 1) individual roots as well as the root system as a whole adapt to the pattern of water availability to maximize access to water, and that 2) this adaptation increases as water sources become more rare. These results suggest that either plant roots are more sensitive to water gradients than humanmade water sensors by 3-5 orders of magnitude, or they might have developed, like other organisms, mechanisms for water foraging that allow them to find water in the absence of an external gradient in water potential.

The intergenic small non-coding RNA ittA is required for optimal infectivity and tissue tropism in Borrelia burgdorferi.

Post-transcriptional regulation via small regulatory RNAs (sRNAs) has been implicated in diverse regulatory processes in bacteria, including virulence. One class of sRNAs, termed trans-acting sRNAs, can affect the stability and/or the translational efficiency of regulated transcripts. In this study, we utilized a collaborative approach that employed data from infection with the Borrelia burgdorferi Tn library, coupled with Tn-seq, together with borrelial sRNA and total RNA transcriptomes, to identify an intergenic trans-acting sRNA, which we designate here as ittA for infectivity-associated and tissue-tropic sRNA locus A. The genetic inactivation of ittA resulted in a significant attenuation in infectivity, with decreased spirochetal load in ear, heart, skin and joint tissues. In addition, the ittA mutant did not disseminate to peripheral skin sites or heart tissue, suggesting a role for ittA in regulating a tissue-tropic response. RNA-Seq analysis determined that 19 transcripts were differentially expressed in the ittA mutant relative to its genetic parent, including vraA, bba66, ospD and oms28 (bba74). Subsequent proteomic analyses also showed a significant decrease of OspD and Oms28 (BBA74) proteins. To our knowledge this is the first documented intergenic sRNA that alters the infectivity potential of B. burgdorferi.

Niemann-Pick C1 Heterogeneity of Bat Cells Controls Filovirus Tropism.

Fruit bats are suspected to be natural hosts of filoviruses, including Ebola virus (EBOV) and Marburg virus (MARV). Interestingly, however, previous studies suggest that these viruses have different tropisms depending on the bat species. Here, we show a molecular basis underlying the host-range restriction of filoviruses. We find that bat-derived cell lines FBKT1 and ZFBK13-76E show preferential susceptibility to EBOV and MARV, respectively, whereas the other bat cell lines tested are similarly infected with both viruses. In FBKT1 and ZFBK13-76E, unique amino acid (aa) sequences are found in the Niemann-Pick C1 (NPC1) protein, one of the cellular receptors interacting with the filovirus glycoprotein (GP). These aa residues, as well as a few aa differences between EBOV and MARV GPs, are crucial for the differential susceptibility to filoviruses. Taken together, our findings indicate that the heterogeneity of bat NPC1 orthologs is an important factor controlling filovirus species-specific host tropism.

Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells from the Tasmanian Devil (Sarcophilus harrisii) Express Immunomodulatory Factors and a Tropism Toward Devil Facial Tumor Cells.

Marsupials have long attracted scientific interest because of their unique biological features and their position in mammalian evolution. Mesenchymal stem cells (MSCs) are of considerable research interest in translational medicine due to their immunomodulatory, anti-inflammatory, and regenerative properties. MSCs have been harvested from various tissues in numerous eutherian species; however, there are no descriptions of MSCs derived from a marsupial. In this study, we have generated Tasmanian devil (Sarcophilus harrisii) MSCs from devil induced pluripotent stem cells (iPSCs), thus providing an unlimited source of devil MSCs and circumventing the need to harvest tissues from live animals. Devil iPSCs were differentiated into MSCs (iMSCs) through both embryoid body formation assays (EB-iMSCs) and through inhibition of the transforming growth factor beta/activin signaling pathway (SB-iMSCs). Both EB-iMSCs and SB-iMSCs are highly proliferative and express the MSC-specific surface proteins CD73, CD90, and CD105, in addition to the pluripotency transcription factors OCT4/POU5F1, SOX2, and NANOG. Expression of the marsupial pluripotency factor POU5F3, a paralogue of OCT4/POU5F1, is significantly reduced in association with the transition from pluripotency to multipotency. Devil iMSCs readily differentiate along the adipogenic, osteogenic, and chondrogenic pathways in vitro, confirming their trilineage differentiation potential. Importantly, in vitro teratoma assays confirmed their multipotency, rather than pluripotency, since the iMSCs only formed derivatives of the mesodermal germ layer. Devil iMSCs show a tropism toward medium conditioned by devil facial tumor cells and express a range of immunomodulatory and anti-inflammatory factors. Therefore, devil iMSCs will be a valuable tool for further studies on marsupial biology and may facilitate the development of an MSC-based treatment strategy against Devil Facial Tumor Disease.

Genetic variability of porcine pegivirus in pigs from Europe and China and insights into tissue tropism.

Pegiviruses belong to the family Flaviviridae and have been found in humans and other mammalian species. To date eleven different pegivirus species (Pegivirus A-K) have been described. However, little is known about the tissue tropism and replication of pegiviruses. In 2016, a so far unknown porcine pegivirus (PPgV, Pegivirus K) was described and persistent infection in the host, similar to human pegivirus, was reported. In this study, qRT-PCR, phylogenetic analyses and fluorescence in situ hybridization (FISH) were implemented to detect and quantify PPgV genome content in serum samples from domestic pigs from Europe and Asia, in tissue and peripheral blood mononuclear cell (PBMC) samples and wild boar serum samples from Germany. PPgV was detectable in 2.7% of investigated domestic pigs from Europe and China (viral genome load 2.4 × 102 to 2.0 × 106 PPgV copies/ml), while all wild boar samples were tested negative. Phylogenetic analyses revealed pairwise nucleotide identities >90% among PPgVs. Finally, PPgV was detected in liver, thymus and PBMCs by qRT-PCR and FISH, suggesting liver- and lymphotropism. Taken together, this study provides first insights into the tissue tropism of PPgV and shows its distribution and genetic variability in Europe and China.

Modulation of Sialic Acid Dependence Influences the Central Nervous System Transduction Profile of Adeno-associated Viruses.

Central nervous system (CNS) transduction by systemically administered recombinant adeno-associated viral (AAV) vectors requires crossing the blood-brain barrier (BBB). We recently mapped a structural footprint on the AAVrh.10 capsid, which, when grafted onto the AAV1 capsid (AAV1RX), enables viral transport across the BBB; however, the underlying mechanisms remain unknown. Here, we establish through structural modeling that this footprint overlaps in part the sialic acid (SIA) footprint on AAV1. We hypothesized that altered SIA-capsid interactions may influence the ability of AAV1RX to transduce the CNS. Using AAV1 variants with altered SIA footprints, we map functional attributes of these capsids to their relative SIA dependence. Specifically, capsids with ablated SIA binding can penetrate and transduce the CNS with low to moderate efficiency. In contrast, AAV1 shows strong SIA dependency and does not transduce the CNS after systemic administration and, instead, transduces the vasculature and the liver. The AAV1RX variant, which shows an intermediate SIA binding phenotype, effectively enters the brain parenchyma and transduces neurons at levels comparable to the level of AAVrh.10. In corollary, the reciprocal swap of the AAV1RX footprint onto AAVrh.10 (AAVRX1) attenuated CNS transduction relative to that of AAVrh.10. We conclude that the composition of residues within the capsid variable region 1 (VR1) of AAV1 and AAVrh.10 profoundly influences tropism, with altered SIA interactions playing a partial role in this phenotype. Further, we postulate a Goldilocks model, wherein optimal glycan interactions can influence the CNS transduction profile of AAV capsids.IMPORTANCE Understanding how viruses cross the blood-brain barrier can provide insight into new approaches to block infection by pathogens or the ability to exploit these pathways for designing new recombinant viral vectors for gene therapy. In this regard, modulation of virus-carbohydrate interactions by mutating the virion shell can influence the ability of recombinant viruses to cross the vascular barrier, enter the brain, and enable efficient gene transfer to neurons.

Bovine endometrial MSC: mesenchymal to epithelial transition during luteolysis and tropism to implantation niche for immunomodulation.

BACKGROUND: The uterus is a histologically dynamic organ, and the mechanisms coordinating its regeneration during the oestrous cycle and implantation are poorly understood. The aim of this study was to isolate, immortalize and characterize bovine endometrial mesenchymal stem cell (eMSC) lines from different oestrous cycle stages (embryo in the oviduct, embryo in the uterus or absence of embryo) and examine their migratory and immunomodulatory properties in an inflammatory or implantation-like environment, as well as possible changes in cell transdifferentiation. METHODS: eMSCs were isolated and analysed in terms of morphological features, expression of cell surface and intracellular markers of pluripotency, inmunocytochemical analyses, alkaline phosphatase activity, proliferation and osteogenic or chondrogenic differentiation capacities, as well as their ability to migrate in response to inflammatory (TNF-α or IL-1ß) or implantation (IFN-τ) cytokines and their immunomodulatory effect in the proliferation of T cells. RESULTS: All eMSCs showed MSC properties such as adherence to plastic, high proliferative capacity, expression of CD44 and vimentin, undetectable expression of CD34 or MHCII, positivity for Pou5F1 and alkaline phosphatase activity. In the absence of an embryo, eMSC showed an apparent mesenchymal to epithelial transition state. eMSC during the entire oestrous cycle differentiated to osteogenic or chondrogenic lineages, showed the ability to suppress T cell proliferation and showed migratory capacity towards pro-inflammatory signal, while responded with a block in their migration to the embryo-derived pregnancy signal. CONCLUSION: This study describes for the first time the isolation, immortalization and characterization of bovine mesenchymal stem cell lines from different oestrous cycle stages, with a clear mesenchymal pattern and immunomodulatory properties. Our study also reports that the migratory capacity of the eMSC was increased towards an inflammatory niche but was reduced in response to the expression of implantation cytokine by the embryo. The combination of both signals (pro-inflammatory and implantation) would ensure the retention of eMSC in case of pregnancy, to ensure the immunomodulation necessary in the mother for embryo survival. In addition, in the absence of an embryo, eMSC showed an apparent mesenchymal to epithelial transition state.