Circulation of small ruminant lentivirus in endangered goat and sheep breeds of Southern Italy.

According to the Domestic Animal Diversity Information System (DAD-IS) of the FAO, Italy has one of the largest numbers of local small ruminant breeds among European countries. In Southern Italy, namely the Campania Region, Bagnolese and Laticauda sheep breeds and Cilentana goat breeds are considered endangered according to the DAD-IS. Conservation of endangered animal breeds is a goal of the European Union (EU). However, the role of infectious diseases as risk factors for endangered breeds has rarely been considered. Small ruminant lentiviruses (SRLV) infect sheep and goats, causing slow-progressive, persistent, and debilitating diseases that can lead to animal death and productivity loss. In this study, we investigated the presence of SRLV in Bagnolese, Laticauda, and Cilentana breeds using a commercial ELISA in parallel with an in-house ELISA. The results of the two tests were in good agreement (Cohen Kappa 0.84, 95 % CI = 0.76-0.93). Discrepancies between the two tests were resolved using western blotting. In total, 430 samples were tested (248 Bagnolese, 125 Laticauda, and 57 Cilentana). The apparent prevalence rates were 12.5 %, 6.4 %, and 1.7 % in Bagnolese, Laticauda, and Cilentana, respectively. In the molecular analysis of 11 proviral partial sequences, subtypes B2 and A24 were identified in two Bagnolese herds. Owing to the beneficial role of sheep and goat breeding in marginal areas, it is important to screen the entire population and implement control/eradication of SRLV infections in conjunction with each conservation program.

Investigation, management and control of a maedi outbreak in Norway in 2019-2020.

BACKGROUND: Visna-maedi is a notifiable disease in Norway, and eliminating the disease is a national goal. The import of sheep into Norway is very limited, and strict regulations apply to the movement of small ruminants between flocks and within defined geographical regions. Several outbreaks have occurred in the last 50 years, and the most recent before 2019 occurred in Trøndelag county in Central Norway in 2002. A national surveillance programme for small ruminant lentivirus infection exists since 2003. RESULTS: In 2019, the national surveillance programme detected seropositive animals for small ruminant lentivirus in a sheep flock in Trøndelag. Based on the result of polymerase chain reaction analysis and histopathological findings, the Norwegian Food Safety Authority concluded the diagnosis of maedi. Further investigations detected maedi in eight additional sheep flocks in the same county. The flocks were placed under restrictions, and the authorities also imposed restrictions on 82 contact flocks. Sequencing of partial gag genes indicated that the virus in the current outbreak was related to the small ruminant lentivirus detected in the same area between 2002 and 2005. CONCLUSIONS: The outbreak investigation shows the need for sensitive and specific diagnostic methods, and an improved and more targeted surveillance strategy. It also demonstrates the risk of disease spreading between flocks through animal movements, and highlights the importance of biosecurity and structured livestock trade. In addition to allowing livestock trade only from flocks documented free from maedi, it may be necessary to monitor sheep flocks over many years, when aiming to eliminate maedi from the Norwegian sheep population.

Transcription Factor-Mediated Generation of Dopaminergic Neurons from Human iPSCs-A Comparison of Methods.

Dopaminergic neurons are the predominant brain cells affected in Parkinson's disease. With the limited availability of live human brain dopaminergic neurons to study pathological mechanisms of Parkinson's disease, dopaminergic neurons have been generated from human-skin-cell-derived induced pluripotent stem cells. Originally, induced pluripotent stem-cell-derived dopaminergic neurons were generated using small molecules. These neurons took more than two months to mature. However, the transcription-factor-mediated differentiation of induced pluripotent stem cells has revealed quicker and cheaper methods to generate dopaminergic neurons. In this study, we compared and contrasted three protocols to generate induced pluripotent stem-cell-derived dopaminergic neurons using transcription-factor-mediated directed differentiation. We deviated from the established protocols using lentivirus transduction to stably integrate different transcription factors into the AAVS1 safe harbour locus of induced pluripotent stem cells. We used different media compositions to generate more than 90% of neurons in the culture, out of which more than 85% of the neurons were dopaminergic neurons within three weeks. Therefore, from our comparative study, we reveal that a combination of transcription factors along with small molecule treatment may be required to generate a pure population of human dopaminergic neurons.

Lentiviral vector determinants of anion-exchange chromatography elution heterogeneity.

The demand for Lentiviral Vector (LV) drug substance is increasing. However, primary capture using convective anion-exchange chromatography remains a significant manufacturing challenge. This stems from a poor understanding of the complex adsorption behaviors linked to LVs intricate and variable structure, such as high binding heterogeneity which is typically characterized by a gradient elution profile consisting of two peaks. Understanding which LV structural components drive these phenomena is therefore crucial for rational process design. This work identifies the key LV envelope components responsible for binding to quaternary-amine membrane adsorbents. Eliminating the pseudotype protein (Vesicular Stomatitis Virus G glycoprotein [VSV-G]) did not impact the heterogenous two-peak elution profile, suggesting it is not a major binding species. Digestion of envelope glycosaminoglycans (GAGs), present on proteoglycans, leads to a dramatic reduction in the proportion of vector eluted in peak 2, decreasing from 50% to 3.1%, and a threefold increase in peak 1 maximum. Data from reinjection experiments point towards interparticle envelope heterogeneity from discrete LV populations, where the two-peak profile emerges from a subpopulation of LVs interacting via highly charged GAGs (peak 2) along with a weaker binding population likely interacting through the phospholipid membrane and envelope protein (peak 1).

Genetic alteration of SJ293TS cells and modification of serum-free media enhances lentiviral vector production.

Successful cell and gene therapy clinical trials have resulted in the US Food and Drug Administration and European Medicines Agency approving their use for treatment of patients with certain types of cancers and monogenetic diseases. These novel therapies, which rely heavily on lentiviral vectors to deliver therapeutic transgenes to patient cells, have driven additional investigations, increasing demand for both pre-clinical and current Good Manufacturing Practices-grade viral vectors. To better support novel studies by improving current production methods, we report the development of a genetically modified HEK293T-based cell line that is null for expression of both Protein Kinase R and Beta-2 microglobulin and grows in suspension using serum-free media, SJ293TS-DPB. Absence of Protein Kinase R increased anti-sense lentiviral vector titers by more than 7-fold, while absence of Beta-2 microglobulin, a key component of major histocompatibility complex class I molecules, has been reported to reduce the immunogenicity of lentiviral particles. Furthermore, we describe an improved methodology for culturing SJ293TS-DPB that facilitates expansion, reduces handling, and increases titers by 2-fold compared with previous methods. SJ293TS-DPB stably produced lentiviral vectors for over 4 months and generated lentiviral vectors that efficiently transduce healthy human donor T cells and CD34+ hematopoietic stem cells.

Prospects and limitations of cumate-inducible lentivirus as a tool for investigating VEGF-A-mediated pathology in diabetic retinopathy.

Diabetic retinopathy (DR) is a multifactorial disease displaying vascular-associated pathologies, including vascular leakage and neovascularization, ultimately leading to visual impairment. However, animal models accurately reflecting these pathologies are lacking. Vascular endothelial growth factor A (VEGF-A) is an important factor in the development of micro- and macro-vascular pathology in DR. In this study, we evaluated the feasibility of using a cumate-inducible lentivirus (LV) mediated expression of vegf-a to understand DR pathology in vitro and in vivo. Retinal pigment epithelial cells (ARPE-19) were transduced with cumate-inducible LV expressing vegf-a, with subsequent analysis of vegf-a expression and its impact on cell proliferation, viability, motility, and permeability. Cumate tolerability in adult Wistar rat eyes was assessed as an initial step towards a potential DR animal model development, by administering cumate via intravitreal injections (IVT) and evaluating consequent effects by spectral domain optical coherence tomography (SD-OCT), flash electroretinography (fERG), ophthalmic examination (OE), and immunohistochemistry. Transduction of ARPE-19 cells with cumate-inducible LV resulted in ~ 2.5-fold increase in vegf-a mRNA and ~ threefold increase in VEGF-A protein secretion. Transduced cells displayed enhanced cell proliferation, viability, permeability, and migration in tube-like structures. However, IVT cumate injections led to apparent retinal toxicity, manifesting as retinal layer abnormalities, haemorrhage, vitreous opacities, and significant reductions in a- and b-wave amplitudes, along with increased microglial activation and reactive gliosis. In summary, while cumate-inducible LV-mediated vegf-a expression is valuable for in vitro mechanistic studies in cellular drug discovery, its use is not a feasible approach to model DR in in vivo studies due to cumate-induced retinal toxicity.

Construction and immunogenicity of SARS-CoV-2 virus-like particle expressed by recombinant baculovirus BacMam.

The pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve to give rise to variants of concern that can escape vaccine-induced immunity. As such, more effective vaccines are urgently needed. In this study, we evaluated virus-like particle (VLP) as a vaccine platform for SARS-CoV-2. The spike, envelope, and membrane proteins of the SARS-CoV-2 Wuhan strain were expressed by a single recombinant baculovirus BacMam and assembled into VLPs in cell culture. The morphology and size of the SARS-CoV-2 VLP as shown by transmission electron microscopy were similar to the authentic SARS-CoV-2 virus particle. In a mouse trial, two intramuscular immunizations of the VLP BacMam with no adjuvant elicited spike-specific binding antibodies in both sera and bronchoalveolar lavage fluids. Importantly, BacMam VLP-vaccinated mouse sera showed neutralization activity against SARS-CoV-2 spike pseudotyped lentivirus. Our results indicated that the SARS-CoV-2 VLP BacMam stimulated spike-specific immune responses with neutralization activity. IMPORTANCE: Although existing vaccines have significantly mitigated the impact of the COVID-19 pandemic, none of the vaccines can induce sterilizing immunity. The spike protein is the main component of all approved vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) due primarily to its ability to induce neutralizing antibodies. The conformation of the spike protein in the vaccine formulation should be critical for the efficacy of a vaccine. By way of closely resembling the authentic virions, virus-like particles (VLPs) should render the spike protein in its natural conformation. To this end, we utilized the baculovirus vector, BacMam, to express virus-like particles consisting of the spike, membrane, and envelope proteins of SARS-CoV-2. We demonstrated the immunogenicity of our VLP vaccine with neutralizing activity. Our data warrant further evaluation of the virus-like particles as a vaccine candidate in protecting against virus challenges.

Augmenting the landscape of chimeric antigen receptor T-cell therapy.

INTRODUCTION: The inception of recombinant DNA technology and live cell genomic alteration have paved the path for the excellence of cell and gene therapies and often provided the first curative treatment for many indications. The approval of the first Chimeric Antigen Receptor (CAR) T-cell therapy was one of the breakthrough innovations that became the headline in 2017. Currently, the therapy is primarily restricted to a few nations, and the market is growing at a CAGR (current annual growth rate) of 11.6% (2022-2032), as opposed to the established bio-therapeutic market at a CAGR of 15.9% (2023-2030). The limited technology democratization is attributed to its autologous nature, lack of awareness, therapy inclusion criteria, high infrastructure cost, trained personnel, complex manufacturing processes, regulatory challenges, recurrence of the disease, and long-term follow-ups. AREAS COVERED: This review discusses the vision and strategies focusing on the CAR T-cell therapy democratization with mitigation plans. Further, it also covers the strategies to leverage the mRNA-based CAR T platform for building an ecosystem to ensure availability, accessibility, and affordability to the community. EXPERT OPINION: mRNA-guided CAR T cell therapy is a rapidly growing area wherein a collaborative approach among the stakeholders is needed for its success.

Fate control engagement augments NK cell responses in LV/hu-IL-12 transduced sarcoma.

INTRODUCTION: NK cells are an untapped resource for cancer therapy. Sarcomas transduced with lentiviruses to express human IL-12 are only cleared in mice bearing mature human NK cells. However, systemic inflammation limits IL-12 utilization. Fate control a.k.a. "suicide mechanisms" regulate unchecked systemic inflammation caused by cellular immunotherapies. Despite increasing utilization, there remains limited data on immune consequences or tumor-directed effects of fate control. OBJECTIVES: We sought to engage the mutant thymidylate kinase (mTMPK) metabolic fate control system to regulate systemic inflammation and assess the impact on NK cell effector functions. METHODS: Primary human sarcoma short-passage samples and cell lines were transduced with LV/hu-IL-12_mTMPK engineering expression of IL-12 and an AZT-associated fate control enzyme. We assessed transduced sarcoma responses to AZT engagement and subsequent modulation of NK cell functions as measured by inflammatory cytokine production and cytotoxicity. RESULTS: AZT administration to transduced (LV/hu-IL-12_mTMPK) short-passage primary human sarcomas and human Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma cell lines, abrogated the robust expression of human IL-12. Fate control activation elicited a specific dose-dependent cytotoxic effect measured by metabolic activity (WST-1) and cell death (Incucyte). NK effector functions of IFN-γ and cytotoxic granule release were significantly augmented despite IL-12 abrogation. This correlated with preferentially induced expression of NK cell activation ligands. CONCLUSIONS: mTMPK fate control engagement terminates transduced sarcoma IL-12 production and triggers cell death, but also augments an NK cell-mediated response coinciding with metabolic stress activating surface ligand induction. Fate control engagement could offer a novel immune activation method for NK cell-mediated cancer clearance.

Analysis of Surface Expression of NMDAR Subunits in Primary Hippocampal Neurons.

The expression and activity of ionotropic glutamate receptors control signal transduction at the excitatory synapses in the CNS. The NMDAR comprises two obligatory GluN1 subunits and two GluN2 or GluN3 subunits in different combinations. Each GluN subunit consists of four domains: the extracellular amino-terminal and agonist-binding domains, the transmembrane domain, and the intracellular C-terminal domain (CTD). The CTD interaction with various classes of intracellular proteins is critical for trafficking and synaptic localization of NMDARs. Amino acid mutations or the inclusion of premature stop codons in the CTD could contribute to the emergence of neurodevelopmental and neuropsychiatric disorders. Here, we describe the method of preparing primary hippocampal neurons and lentiviral particles expressing GluN subunits that can be used as a model to study cell surface expression and synaptic localization of NMDARs. We also show a simple method of fluorescence immunostaining of eGFP-tagged GluN2 subunits and subsequent microscopy technique and image analysis to study the effects of disease-associated mutations in the CTDs of GluN2A and GluN2B subunits.

Modified lentiviral globin gene therapy for pediatric ß0/ß0 transfusion-dependent ß-thalassemia: A single-center, single-arm pilot trial.

ß0/ß0 thalassemia is the most severe type of transfusion-dependent ß-thalassemia (TDT) and is still a challenge facing lentiviral gene therapy. Here, we report the interim analysis of a single-center, single-arm pilot trial (NCT05015920) evaluating the safety and efficacy of a ß-globin expression-optimized and insulator-engineered lentivirus-modified cell product (BD211) in ß0/ß0 TDT. Two female children were enrolled, infused with BD211, and followed up for an average of 25.5 months. Engraftment of genetically modified hematopoietic stem and progenitor cells was successful and sustained in both patients. No unexpected safety issues occurred during conditioning or after infusion. Both patients achieved transfusion independence for over 22 months. The treatment extended the lifespan of red blood cells by over 42 days. Single-cell DNA/RNA-sequencing analysis of the dynamic changes of gene-modified cells, transgene expression, and oncogene activation showed no notable adverse effects. Optimized lentiviral gene therapy may safely and effectively treat all ß-thalassemia.

Challenges in the analysis of pharmaceutical lentiviral vector products by orthogonal and complementary physical (nano)particle characterization techniques.

Lentiviral vectors (LVVs) are used as a starting material to generate chimeric antigen receptor (CAR) T cells. Therefore, LVVs need to be carefully analyzed to ensure safety, quality, and potency of the final product. We evaluated orthogonal and complementary analytical techniques for their suitability to characterize particulate matter (impurities and LVVs) in pharmaceutical LVV materials at development stage derived from suspension and adherent manufacturing processes. Microfluidic resistive pulse sensing (MRPS) with additional manual data fitting enabled the assessment of mode diameters for particles in the expected LVV size range in material from adherent production. LVV material from a suspension process, however, contained substantial amounts of particulate impurities which blocked MRPS cartridges. Sedimentation-velocity analytical ultracentrifugation (SV-AUC) resolved the LVV peak in material from adherent production well, whereas in more polydisperse samples from suspension production, presence of particulate impurities masked a potential signal assignable to LVVs. In interferometric light microscopy (ILM) and nanoparticle tracking analysis (NTA), lower size detection limits close to âˆ¼ 70 nm resulted in an apparent peak in particle size distributions at the expected size for LVVs emphasizing the need to interpret these data with care. Interpretation of data from dynamic light scattering (DLS) was limited by insufficient size resolution and sample polydispersity. In conclusion, the analysis of LVV products manufactured at pharmaceutical scale with current state-of-the-art physical (nano)particle characterization techniques was challenging due to the presence of particulate impurities of heterogeneous size. Among the evaluated techniques, MRPS and SV-AUC were most promising yielding acceptable results at least for material from adherent production.

Targeting the long non-coding RNA MIAT for the treatment of fibroids in an animal model.

Our previous studies indicated that there is overexpression of MIAT in fibroids and MIAT is a sponge for the miR-29 family in these tumors. The objective of the present study was to determine if the knockdown of MIAT in fibroid xenografts will increase miR-29 levels and reduce the expression of genes targeted by this miRNA such as collagen and cell cycle regulatory proteins in a mouse model for fibroids. Ovariectomized CB-17 SCID/Beige mice bearing estrogen/progesterone pellets were implanted subcutaneously in the flank with equal weight of fibroid explants which had been transduced by lentivirus for either control (empty vector) or MIAT knockdown for four weeks (n=7). Knockdown of MIAT in fibroid xenografts resulted in a 30% reduction of tumor weight and a marked increase in miR-29a, -b, and -c levels in the xenografts. There was reduced cell proliferation and expression of cell cycle regulatory genes CCND1, CDK2, and E2F1 and no significant changes in apoptosis. The xenografts with MIAT knockdown expressed lower mRNA and protein levels of FN1, COL3A1, and TGF-ß3, and total collagen protein. Targeting MIAT, which sponges the pro-fibrotic miR-29 family, is an effective therapy for fibroids by reducing cell proliferation and thereby, tumor growth and accumulation of ECM, which is a hallmark of these benign gynecologic tumors.

Development of a Bead-Based Multiplex Fluorescent Immunoassay to Detect Antibodies against Maedi-Visna Virus in Sheep.

The Maedi-visna virus (MVV) causes a persistent infection in small ruminants, and its high genetic heterogeneity affects the performance of diagnostic tests when used in different populations. Therefore, the aim of this study was to develop a bead-based multiplex immunoassay tailored to detect antibodies against a Norwegian MVV strain. We used tissue samples from 14 PCR-positive sheep from a recent MVV outbreak in Norway to sequence the viral strain and produced recombinant antigens based on sequences from one animal. The assay included commercial TM-A and recombinant Norwegian p25, p16-25 and SU5 antigens. Cut-off values for each antigen were determined using receiver operating characteristic curves on 40 ELISA-negative and 67 ELISA-positive samples from the outbreak. The intraplate and interplate repeatability were investigated by testing a quadruplicate of five samples over three days, while the analytical sensitivity (aSe) and specificity (aSp) were measured in comparison to a commercial ELISA. The repeatability showed a coefficient of variation below 15% for most positive samples. The aSe was equal or higher for the multiplex assay than the ELISA, and the aSp of each antigen was 91.7, 93.3, 95.0 and 93.3% for p25, p16-25, SU5 and TM-A, respectively. The assay shows promising results; however, further evaluations of diagnostic characteristics are necessary before implementation in the Norwegian surveillance programme.

Comparing chemical transfection, electroporation, and lentiviral vector transduction to achieve optimal transfection conditions in the Vero cell line.

BACKGROUND: Transfection is an important analytical method for studying gene expression in the cellular environment. There are some barriers to efficient DNA transfection in host cells, including circumventing the plasma membrane, escaping endosomal compartmentalization, autophagy, immune sensing pathways, and translocating the nuclear envelope. Therefore, it would be very useful to introduce an optimum transfection approach to achieve a high transfection efficiency in the Vero cell line. The aim of this study was to compare various transfection techniques and introduce a highly efficient method for gene delivery in Vero cells. METHODS: In the current study, three transfection methods were used, including chemical transfection, electroporation, and lentiviral vector transduction, to obtain the optimum transfection conditions in the Vero cell line. Vero cells were cultured and transfected with chemical transfection reagents, electroporation, or HIV-1-based lentivectors under different experimental conditions. Transfection efficiency was assessed using flow cytometry and fluorescence microscopy to detect GFP-positive cells. RESULTS: Among the tested methods, TurboFect™ chemical transfection exhibited the highest efficiency. Optimal transfection conditions were achieved using 1 µg DNA and 4 µL TurboFect™ in 6 × 104 Vero cells. CONCLUSION: TurboFect™, a cationic polymer transfection reagent, demonstrated superior transfection efficiency in Vero cells compared with electroporation and lentivirus particles, and is the optimal choice for chemical transfection in the Vero cell line.

A lentivirus-vectored feline erythropoietin gene therapy strategy in tissue culture and rodent models for the potential treatment of chronic renal disease-associated anemia.

OBJECTIVE: The aim of this study was to assess the efficacy and safety of a third-generation lentivirus-based vector encoding the feline erythropoietin (EPO) (feEPO) gene in vitro and in rodent models in vivo. This vector incorporates a genetic mechanism to facilitate the termination of the therapeutic effect in the event of supraphysiologic polycythemia, the herpes simplex virus thymidine kinase (HSV-TK) "suicide gene." ANIMALS: CFRK cells and replication-defective lentiviral vectors encoding feEPO were used for in vitro experiments. Eight Fischer rats were enrolled in the pilot in vivo study, 24 EPO-deficient mice were used in the initial mouse study, and 15 EPO-deficient mice were enrolled in the final mouse study. METHODS: Efficacy of a third-generation lentivirus encoding feEPO was determined in vitro using western blot assays. Subsequently, in a series of rodent experiments, animals were administered the viral vector in progressively increasing inoculation doses with serial measurements of blood packed cell volume (PCV) over time. RESULTS: We documented production of feEPO protein in transduced CRFK cells with subsequent cessation of production when treated with the HSV-TK substrate ganciclovir. In vivo, we demonstrated variably persistent elevated PCV values in treated rats and mice with eventual return to baseline values over time. CLINICAL RELEVANCE: These results provide justification for a lentiviral gene therapy approach to the treatment of nonregenerative anemia associated with chronic renal disease in cats.

Dual-layer conduit containing VEGF-A - Transfected Schwann cells promotes peripheral nerve regeneration via angiogenesis.

Peripheral nerve injuries (PNIs) can cause neuropathies and significantly affect the patient's quality of life. Autograft transplantation is the gold standard for conventional treatment; however, its application is limited by nerve unavailability, size mismatch, and local tissue adhesion. Tissue engineering, such as nerve guidance conduits, is an alternative and promising strategy to guide nerve regeneration for peripheral nerve repair; however, only a few conduits could reach the high repair efficiency of autografts. The healing process of PNI is frequently accompanied by not only axonal and myelination regeneration but also angiogenesis, which initializes nerve regeneration through vascular endothelial growth factor A (VEGF-A). In this study, a composite nerve conduit with a poly (lactic-co-glycolic acid) (PLGA) hollow tube as the outer layer and gelatin methacryloyl (GelMA) encapsulated with VEGF-A transfected Schwann cells (SCs) as the inner layer was established to evaluate its promising ability for peripheral nerve repair. A rat model of peripheral nerve defect was used to examine the efficiency of PLGA/GelMA-SC (VA) conduits, whereas autograft, PLGA, PLGA/GelMA, and PLGA/GelMA-SC (NC) were used as controls. VEGF-A-transfected SCs can provide a stable source for VEGF-A secretion. Furthermore, encapsulation in GelMA cannot only promote proliferation and tube formation of human umbilical vein endothelial cells but also enhance dorsal root ganglia and neuronal cell extension. Previous animal studies have demonstrated that the regenerative effects of PLGA/GelMA-SC (VA) nerve conduit were similar to those of autografts and much better than those of other conduits. These findings indicate that combination of VEGF-A-overexpressing SCs and PLGA/GelMA conduit-guided peripheral nerve repair provides a promising method that enhances angiogenesis and regeneration during nerve repair. STATEMENT OF SIGNIFICANCE: Nerve guidance conduits shows promise for peripheral nerve repair, while achieving the repair efficiency of autografts remains a challenge. In this study, a composite nerve conduit with a PLGA hollow tube as the outer layer and gelatin methacryloyl (GelMA) encapsulated with vascular endothelial growth factor A (VEGF-A)-transfected Schwann cells (SCs) as the inner layer was established to evaluate its potential ability for peripheral nerve repair. This approach preserves growth factor bioactivity and enhances material properties. GelMA insertion promotes Schwann cell proliferation and morphology extension. Moreover, transfected SCs serve as a stable VEGF-A source and fostering angiogenesis. This study offers a method preserving growth factor efficacy and safeguarding SCs, providing a comprehensive solution for enhanced angiogenesis and nerve regeneration.

Enhancing Endogenous Hyaluronic Acid in Osteoarthritic Joints with an Anti-Inflammatory Supramolecular Nanofiber Hydrogel Delivering HAS2 Lentivirus.

Osteoarthritis (OA) management remains challenging because of its intricate pathogenesis. Intra-articular injections of drugs, such as glucocorticoids and hyaluronic acid (HA), have certain limitations, including the risk of joint infection, pain, and swelling. Hydrogel-based therapeutic strategies have attracted considerable attention because of their enormous therapeutic potential. Herein, a supramolecular nanofiber hydrogel is developed using dexamethasone sodium phosphate (DexP) as a vector to deliver lentivirus-encoding hyaluronan synthase 2 (HAS2) (HAS2@DexP-Gel). During hydrogel degradation, HAS2 lentivirus and DexP molecules are slowly released. Intra-articular injection of HAS2@DexP-Gel promotes endogenous HA production and suppresses synovial inflammation. Additionally, HAS2@DexP-Gel reduces subchondral bone resorption in the anterior cruciate ligament transection-induced OA mice, attenuates cartilage degeneration, and delays OA progression. HAS2@DexP-Gel exhibited good biocompatibility both in vitro and in vivo. The therapeutic mechanisms of the HAS2@DexP-Gel are investigated using single-cell RNA sequencing. HAS2@DexP-Gel optimizes the microenvironment of the synovial tissue by modulating the proportion of synovial cell subpopulations and regulating the interactions between synovial fibroblasts and macrophages. The innovative nanofiber hydrogel, HAS2@DexP-Gel, effectively enhances endogenous HA production while reducing synovial inflammation. This comprehensive approach holds promise for improving joint function, alleviating pain, and slowing OA progression, thereby providing significant benefits to patients.

Establishing Primary and Stable Cell Lines from Frozen Wing Biopsies for Cellular, Physiological, and Genetic Studies in Bats.

Bats stand out among mammalian species for their exceptional traits, including the capacity to navigate through flight and echolocation, conserve energy through torpor/hibernation, harbor a multitude of viruses, exhibit resistance to disease, survive harsh environmental conditions, and demonstrate exceptional longevity compared to other mammals of similar size. In vivo studies of bats can be challenging for several reasons such as ability to locate and capture them in their natural environments, limited accessibility, low sample size, environmental variation, long lifespans, slow reproductive rates, zoonotic disease risks, species protection, and ethical concerns. Thus, establishing alternative laboratory models is crucial for investigating the diverse physiological adaptations observed in bats. Obtaining quality cells from tissues is a critical first step for successful primary cell derivation. However, it is often impractical to collect fresh tissue and process the samples immediately for cell culture due to the resources required for isolating and expanding cells. As a result, frozen tissue is typically the starting resource for bat primary cell derivation. Yet, cells in frozen tissue are usually damaged and represent low integrity and viability. As a result, isolating primary cells from frozen tissues poses a significant challenge. Herein, we present a successfully developed protocol for isolating primary dermal fibroblasts from frozen bat wing biopsies. This protocol marks a significant milestone, as this the first protocol specially focused on fibroblasts isolation from bat frozen tissue. We also describe methods for primary cell characterization, genetic manipulation of primary cells through lentivirus transduction, and the development of stable cell lines. Basic Protocol 1: Bat wing biopsy collection and preservation Support Protocol 1: Blood collection from bat- venipuncture Basic Protocol 2: Isolation of primary fibroblasts from adult bat frozen wing biopsy Support Protocol 2: Maintenance of primary fibroblasts Support Protocol 3: Cell banking and thawing of primary fibroblasts Support Protocol 4: Growth curve and doubling time Support Protocol 5: Lentiviral transduction of bat primary fibroblasts Basic Protocol 3: Bat stable fibroblasts cell lines development Support Protocol 6: Bat fibroblasts validation by immunofluorescence staining Support Protocol 7: Chromosome counting.

Phylogenetic analysis of small ruminant lentiviruses in Mongolian sheep supports an ancient east-west split for the genotype A.

The ovine maedi-visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV) are small ruminant lentiviruses (SRLVs) with striking genetic and structural similarities. The presence of SRLV in Mongolian sheep and goats was serologically demonstrated more than a decade ago; however, the viral genotype remains unknown. In total, 329 blood samples were collected from two sheep breeds (i.e., Khalkha and Sumber) in Tov, Govisumber, Arkhangay, Dornogovi, Zavkhan, and Sukhbaatar provinces, Mongolia. Serological and phylogenetic analyses were performed regardless of any apparent clinical signs, although most of the animals appeared healthy. All sheep in three of the six provinces were seronegative, whereas the seroprevalence in the Tov, Govisumber, and Zavkhan provinces averaged 7.9%. Genomic DNA from seropositive animals was tested using hemi-nested polymerase chain reaction, and sub-genomic SRLV sequences were determined from nine samples. Mongolian SRLV sequences clustered within the divergent subtype A22, which was previously found only in Fertile Crescent regions, including Lebanon, Jordan, and Iran, where the first sheep-domestication (Ovis aries) occurred. According to the phylogenetic analysis, genotype A has two ancestors from the ancient Fertile Crescent: (1) Turkish strains and (2) Iranian, Jordanian, and Lebanese strains. The first ancestor spread westward, whereas the second spread eastward, ultimately reaching Mongolia.