An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis.

SpCas9 and AsCas12a are widely utilized as genome-editing tools in human cells. However, their relatively large size poses a limitation for delivery by cargo-size-limited adeno-associated virus (AAV) vectors. The type V-F Cas12f from Acidibacillus sulfuroxidans is exceptionally compact (422 amino acids) and has been harnessed as a compact genome-editing tool. Here, we developed an approach, combining deep mutational scanning and structure-informed design, to successfully generate two AsCas12f activity-enhanced (enAsCas12f) variants. Remarkably, the enAsCas12f variants exhibited genome-editing activities in human cells comparable with those of SpCas9 and AsCas12a. The cryoelectron microscopy (cryo-EM) structures revealed that the mutations stabilize the dimer formation and reinforce interactions with nucleic acids to enhance their DNA cleavage activities. Moreover, enAsCas12f packaged with partner genes in an all-in-one AAV vector exhibited efficient knock-in/knock-out activities and transcriptional activation in mice. Taken together, enAsCas12f variants could offer a minimal genome-editing platform for in vivo gene therapy.

Advancements in NMN biotherapy and research updates in the field of digestive system diseases.

Nicotinamide mononucleotide (NMN), a crucial intermediate in NAD + synthesis, can rapidly transform into NAD + within the body after ingestion. NMN plays a pivotal role in several important biological processes, including energy metabolism, cellular aging, circadian rhythm regulation, DNA repair, chromatin remodeling, immunity, and inflammation. NMN has emerged as a key focus of research in the fields of biomedicine, health care, and food science. Recent years have witnessed extensive preclinical studies on NMN, offering valuable insights into the pathogenesis of age- and aging-related diseases. Given the sustained global research interest in NMN and the substantial market expectations for the future, here, we comprehensively review the milestones in research on NMN biotherapy over the past 10 years. Additionally, we highlight the current research on NMN in the field of digestive system diseases, identifying existing problems and challenges in the field of NMN research. The overarching aim of this review is to provide references and insights for the further exploration of NMN within the spectrum of digestive system diseases.

Identification of steroid-induced osteonecrosis of the femoral head biomarkers based on immunization and animal experiments.

BACKGROUND: Steroid-induced osteonecrosis of femoral head (SONFH) is a severe health risk, and this study aims to identify immune-related biomarkers and pathways associated with the disease through bioinformatics analysis and animal experiments. METHOD: Using SONFH-related datasets obtained from the GEO database, we performed differential expression analysis and weighted gene co-expression network analysis (WGCNA) to extract SONFH-related genes. A protein-protein interaction (PPI) network was then constructed, and core sub-network genes were identified. Immune cell infiltration and clustering analysis of SONFH samples were performed to assess differences in immune cell populations. WGCNA analysis was used to identify module genes associated with immune cells, and hub genes were identified using machine learning. Internal and external validation along with animal experiments were conducted to confirm the differential expression of hub genes and infiltration of immune cells in SONFH. RESULTS: Differential expression analysis revealed 502 DEGs. WGCNA analysis identified a blue module closely related to SONFH, containing 1928 module genes. Intersection analysis between DEGs and blue module genes resulted in 453 intersecting genes. The PPI network and MCODE module identified 15 key targets enriched in various signaling pathways. Analysis of immune cell infiltration showed statistically significant differences in CD8 + t cells, monocytes, macrophages M2 and neutrophils between SONFH and control samples. Unsupervised clustering classified SONFH samples into two clusters (C1 and C2), which also exhibited significant differences in immune cell infiltration. The hub genes (ICAM1, NR3C1, and IKBKB) were further identified using WGCNA and machine learning analysis. Based on these hub genes, a clinical prediction model was constructed and validated internally and externally. Animal experiments confirmed the upregulation of hub genes in SONFH, with an associated increase in immune cell infiltration. CONCLUSION: This study identified ICAM1, NR3C1, and IKBKB as potential immune-related biomarkers involved in immune cell infiltration of CD8 + t cells, monocytes, macrophages M2, neutrophils and other immune cells in the pathogenesis of SONFH. These biomarkers act through modulation of the chemokine signaling pathway, Toll-like receptor signaling pathway, and other pathways. These findings provide valuable insights into the disease mechanism of SONFH and may aid in future drug development efforts.

Osteoprotective effect of Achyranthes bidentata root extract on osteoporotic rats: a systematic review and meta-analysis.

CONTEXT: Achyranthes bidentata Blume (ABB), a plant of Amaranthaceae family, has been one of the more commonly used phytomedicine remedies for thousands of years, and recent studies have highlighted the efficacy of its extracts in the treatment of osteoporosis. Nonetheless, a thorough analysis of its benefits is currently absent. OBJECTIVE: This meta-analysis assessed the effects of ABB root extract (ABBRE) on osteoporotic rats and provides a rationale for future clinical studies. METHODS: Searches were conducted in seven different Chinese and English databases, and the search period was from their establishment to January 2024. This study was registered in PROSPERO (CRD42023418917). Selected research regarding the ABBRE treatment of osteoporotic rats, and the corresponding research has distinctly reported outcomes, and the data on the bone mineral density (BMD), bone histomorphometrics, biomechanical parameters, and bone biochemical markers of osteoporotic rats can be extracted. RESULTS: Through screening, 11 studies met the eligibility requirements for inclusion, in which 222 animals were studied. The treatment group with ABBRE exhibited increased bone mineral density (standardized mean difference [SMD] = 1.64, 95% CI = 0.52 to 2.77). Based on subgroup analysis, the greatest increase in bone mineral density was observed when the dose of ABBRE was ≤ 400 mg/kg/day and the duration of treatment was ≤ 12 weeks. CONCLUSIONS: ABBRE is a phytomedicine that can effectively promote the enhancement of bone mineral density and ease osteoporosis. It can be developed into a new alternative therapy by conducting experiments and clinical studies on larger samples.

[Mechanism of Shexiang Tongxin Dropping Pills in treating diabetic cardiomyopathy based on network pharmacology and animal experiments].

This study aimed to explore the mechanism of Shexiang Tongxin Dropping Pills(STDP) in treating diabetic cardiomyopathy(DCM) based on network pharmacology, molecular docking, and animal experiments. BATMAN, TCMSP, and GeneCards were searched for the active ingredients and targets of STDP against DCM. STRING and Cytoscape were used to build the protein-protein interaction(PPI) network and "drug-active ingredient-target" network. Gene Ontology(GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analysis of the targets were carried out based on DAVID. The molecular docking of key receptor proteins with corresponding active ingredients was performed using AutoDock Vina. The rat model of DCM was established by a high-fat diet combined with intraperitoneal injection of streptozotocin. Rats were assigned into control, model, low-(20 mg·kg~(-1)) and high-dose(40 mg·kg~(-1)) STDP, and metformin(200 mg·kg~(-1)) groups. After 8 weeks of continuous administration, the cardiac function, myocardial pathological changes, and myocardial collagen fiber deposition of rats in each group were detected by echocardiography, hematoxylin-eosin(HE) staining, and Sirius red staining, respectively. The myocardial hypertrophy was detected by WGA staining. The expression levels of p38 mitogen-activated protein kinase(p38), phosphorylation-p38(p-p38), c-Jun N-terminal kinase(JNK), phosphorylation-JNK(p-JNK), caspase-3, and C-caspase-3 in the myocardial tissue of rats in each group were measured by Western blot. The network pharmacology predicted 199 active ingredients and 1 655 targets of STDP and 463 targets of DCM. One hundred and thirty-four potential targets of STDP for treating DCM were obtained, and the AGE-RAGE signaling pathway in diabetic complications was screened out. Molecular docking results showed that miltirone, dehydromiltirone, and tryptanthrin had strong binding affinity with RAGE. The results of animal experiments confirmed that STDP effectively protected the cardiac function of DCM rats. Compared with the DCM model group, the STDP groups showed significantly down-regulated protein levels of p-p38, p-JNK, and C-caspase-3. To sum up, STDP may protect the cardiac function of DCM rats by regulating the AGE-RAGE signaling pathway.

Bioresorbable Poly (L-Lactic Acid) Flow Diverter Versus Cobalt-Chromium Flow Diverter: In Vitro and In Vivo Analysis.

BACKGROUND: Permanent metallic flow diverter (FD) implantation for treatment of intracranial aneurysms requires antiplatelet therapy for an unclear duration and restricts postprocedural endovascular access. Bioresorbable FDs are being developed as a solution to these issues, but the biological reactions and phenomena induced by bioresorbable FDs have not been compared with those of metallic FDs. METHODS: We have developed a bioresorbable poly (L-lactic acid) FD (PLLA-FD) and compared it with an FD composed of cobalt-chromium and platinum-tungsten (CoCr-FD). FD mechanical performance and in vitro degradation of the PLLA-FD were evaluated. For in vivo testing in a rabbit aneurysm model, FDs were implanted at the aneurysm site and the abdominal aorta in the PLLA-FD group (n=21) and CoCr-FD group (n=15). Aneurysm occlusion rate, branch patency, and thrombus formation within the FD were evaluated at 3, 6, and 12 months. Local inflammation and neointima structure were also evaluated. RESULTS: Mean strut, porosity, and pore density for the PLLA-FD were 41.7 µm, 60%, and 20 pores per mm2, respectively. The proportion of aneurysms exhibiting a neck remnant or complete occlusion did not significantly differ between the groups; however, the complete occlusion rate was significantly higher in the PLLA-FD group (48% versus 13%; P=0.0399). Branch occlusion and thrombus formation within the FD were not observed in either group. In the PLLA-FD group, CD68 immunoreactivity was significantly higher, but neointimal thickness decreased over time and did not significantly differ from that of the CoCr-FD at 12 months. Collagen fibers significantly predominated over elastic fibers in the neointima in the PLLA-FD group. The opposite was observed in the CoCr-FD group. CONCLUSIONS: The PLLA-FD was as effective as the CoCr-FD in this study and is feasible for aneurysm treatment. No morphological or pathological problems were observed with PLLA-FD over a 1-year period.

Non-Contact Irreversible Electroporation in the Esophagus With a Wet Electrode Approach.

Our objective was to develop a technique for performing irreversible electroporation (IRE) of esophageal tumors while mitigating thermal damage to the healthy lumen wall. We investigated noncontact IRE using a wet electrode approach for tumor ablation in a human esophagus with finite element models for electric field distribution, joule heating, thermal flux, and metabolic heat generation. Simulation results indicated the feasibility of tumor ablation in the esophagus using an catheter mounted electrode immersed in diluted saline. The ablation size was clinically relevant, with substantially lesser thermal damage to the healthy esophageal wall when compared to IRE performed by placing a monopolar electrode directly into the tumor. Additional simulations were used to estimate ablation size and penetration during noncontact wet-electrode IRE (wIRE) in the healthy swine esophagus. A novel catheter electrode was manufactured and wIRE evaluated in seven pigs. wIRE was performed by securing the device in the esophagus and using diluted saline to isolate the electrode from the esophageal wall while providing electric contact. Computed tomography and fluoroscopy were performed post-treatment to document acute lumen patency. Animals were sacrificed within four hours following treatment for histologic analysis of the treated esophagus. The procedure was safely completed in all animals; post-treatment imaging revealed intact esophageal lumen. The ablations were visually distinct on gross pathology, demonstrating full thickness, circumferential regions of cell death (3.52 ± 0.89 mm depth). Acute histologic changes were not evident in nerves or extracellular matrix architecture within the treatment site. Catheter directed noncontact IRE is feasible for performing penetrative ablations in the esophagus while avoiding thermal damage.

Treatment of comminuted metaphyseal distal femoral fractures with a micromotion-balancing osteosynthesis: an animal study.

BACKGROUND: Locking plates are commonly used in the treatment of comminuted metaphyseal distal femoral fractures. However, locking plates form a strong structure and promote asymmetrical callus formation, which is not conducive for rapid fracture healing and may increase fracture risk. To overcome this, we designed a micromotion-balancing fixation system based on locking plates. METHODS: Six healthy pigs (Bama miniature pigs) were used to establish a model of bilateral comminuted distal femoral fracture (AO/ASIF: 33-C2). Standard drilling was performed on one of each pig's hind limbs (control group), whereas eccentric drilling was performed on the other hind limb (experimental group). Both femurs were fixed with a 3-hole locking compression plate using 5-mm-diameter screws. At 12 postoperative weeks, all pigs were euthanized and the femurs with compression plates were radiographically examined. The level of fracture healing and loosening/internal fixation failure were recorded. Bone mineral density, number of trabeculae, trabecular morphology, and calcification precipitations were assessed. RESULTS: All pigs survived, and the fractures healed. No complications related to fracture healing, such as infection and internal fixation failure, were noted. The bone mineral density of the near and far cortical calli, number of the near and far cortical callus trabeculae, and difference in bone mineral density between the near and far cortical calli in the experimental group were significantly higher than those in the control group (p < 0.01). However, the difference in the number of trabeculae between the near and far cortical calli was significantly lower in the experimental group than in the control group (p < 0.01). CONCLUSION: This newly designed system provides stable fixation for comminuted distal femoral fracture, increases the overall strain at the fracture site, and balances the strains at the near and far cortices to achieve uniform callus growth and fracture healing.

First Ex Vivo Animal Study of a Biological Heart Valve Prosthesis Sensorized with Intravalvular Impedance.

IntraValvular Impedance (IVI) sensing is an innovative concept for monitoring heart valve prostheses after implant. We recently demonstrated IVI sensing feasible in vitro for biological heart valves (BHVs). In this study, for the first time, we investigate ex vivo the IVI sensing applied to a BHV when it is surrounded by biological tissue, similar to a real implant condition. A commercial model of BHV was sensorized with three miniaturized electrodes embedded in the commissures of the valve leaflets and connected to an external impedance measurement unit. To perform ex vivo animal tests, the sensorized BHV was implanted in the aortic position of an explanted porcine heart, which was connected to a cardiac BioSimulator platform. The IVI signal was recorded in different dynamic cardiac conditions reproduced with the BioSimulator, varying the cardiac cycle rate and the stroke volume. For each condition, the maximum percent variation in the IVI signal was evaluated and compared. The IVI signal was also processed to calculate its first derivative (dIVI/dt), which should reflect the rate of the valve leaflets opening/closing. The results demonstrated that the IVI signal is well detectable when the sensorized BHV is surrounded by biological tissue, maintaining the similar increasing/decreasing trend that was found during in vitro experiments. The signal can also be informative on the rate of valve opening/closing, as indicated by the changes in dIVI/dt in different dynamic cardiac conditions.

Network Analysis and Experimental Verification of the Mechanisms of Hydroxysafflor Yellow A in Ischemic Stroke Following Atherosclerosis.

Hydroxysafflor yellow A (HSYA) is derived from Carthamus tinctorius L. (Honghua in Chinese) and is used to treat cardiovascular and cerebrovascular disease. However, the mechanism by which HSYA treats ischemic stroke following atherosclerosis (ISFA) remains unclear. The targets and pathways of HSYA against ISFA were obtained using network analysis. A total of 3335 potential IFSA-related targets were predicted using the GenCards and Drugbank databases, and a total of 88 potential HSYA-related targets were predicted using the Swiss Target Prediction database. A total of 62 HSYA-related targets against IFSA were obtained. The network was composed of HSYA, 62 targets, and 20 pathways. The top 20 targets were constructed via the protein-protein interaction (PPI) network. Gene Ontology analysis revealed that the targets were involved in signal transduction, protein phosphorylation, the cytoplasm, the plasma membrane, the cytosol, zinc ion binding, ATP binding, protein kinase binding/activity, and enzyme binding. The Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that the pathways were associated with cancer, inflammatory mediator regulation of the transient receptor potential channels, and microRNA in cancer. Additionally, molecular docking indicated that HSYA mainly interacts with five targets, namely interleukin 1 beta (IL-1ß), signal transducer and activator of transcription 3 (STAT3), E1A-binding protein p300 (EP300), protein kinase C alpha (PRKCA), and inhibitor of nuclear factor kappa B kinase subunit beta (IKBKB). In animal experiments, HSYA administration ameliorated the infarct size, neurological deficit score, histopathological changes, carotid intima-media thickness (IMT), and blood lipid level (total cholesterol and triglycerides). Immunochemistry and quantitative PCR showed that HSYA intervention downregulated the expression of STAT3, EP300, PRKCA, and IKBKB, and the enzyme-linked immunoassay showed reduced IL-1ß levels. The findings of this study provide a reference for the development of anti-ISFA drugs.

The influence of high-speed train's transient pressure change on the fetal growth of SD rats.

This research aims to explore the influence of transient pressure fluctuation inside high-speed trains passing throught tunnels on the fetal growth of Sprague - Dawley (SD) rats. A pressure variation simulation system was designed and exposure experiments were performed on SD rats. Forty-eight SD rats are divided into two control groups and two experimental groups, and are then exposed to transient pressure alternation (-1200 Pa ~1200 Pa) from gestation day 0 to gestation day 5 (GD 0-5). Fetal growth and development indicators on GD12 and GD18 between experimental and control groups were compared. Statistical results showed that, compared to the control group, the key indicators in the experimental group, including placental weight, placental diameter, fetal weight, and crown-to-rump length have decreased by 4.77%, 3.38%, 6.20%, and 3.75% respectively on GD18. The findings imply that the pressure fluctuation environment of high-speed trains has potential effects on the fetal growth of SD rats.

[Chrysin alleviates cerebral ischemia-reperfusion injury by inhibiting ferroptosis in rats].

The purpose of this study is to investigate whether chrysin reduces cerebral ischemia-reperfusion injury(CIRI) by inhi-biting ferroptosis in rats. Male SD rats were randomly divided into a sham group, a model group, high-, medium-, and low-dose chrysin groups(200, 100, and 50 mg·kg~(-1)), and a positive drug group(Ginaton, 21.6 mg·kg~(-1)). The CIRI model was induced in rats by transient middle cerebral artery occlusion(tMCAO). The indexes were evaluated and the samples were taken 24 h after the operation. The neurological deficit score was used to detect neurological function. The 2,3,5-triphenyl tetrazolium chloride(TTC) staining was used to detect the cerebral infarction area. Hematoxylin-eosin(HE) staining and Nissl staining were used to observe the morphological structure of brain tissues. Prussian blue staining was used to observe the iron accumulation in the brain. Total iron, lipid pero-xide, and malondialdehyde in serum and brain tissues were detected by biochemical reagents. Real-time quantitative polymerase chain reaction(RT-qPCR), immunohistochemistry, and Western blot were used to detect mRNA and protein expression of solute carrier fa-mily 7 member 11(SLC7A11), transferrin receptor 1(TFR1), glutathione peroxidase 4(GPX4), acyl-CoA synthetase long chain family member 4(ACSL4), and prostaglandin-endoperoxide synthase 2(PTGS2) in brain tissues. Compared with the model group, the groups with drug intervention showed restored neurological function, decreased cerebral infarction rate, and alleviated pathological changes. The low-dose chrysin group was selected as the optimal dosing group. Compared with the model group, the chrysin groups showed reduced content of total iron, lipid peroxide, and malondialdehyde in brain tissues and serum, increased mRNA and protein expression levels of SLC7A11 and GPX4, and decreased mRNA and protein expression levels of TFR1, PTGS2, and ACSL4. Chrysin may regulate iron metabolism via regulating the related targets of ferroptosis and inhibit neuronal ferroptosis induced by CIRI.

Preclinical evaluation of the new EDGE SP 1000 single-port robotic surgical system in gynecology minimal access surgery.

OBJECTIVE: The aim of this study is to evaluate the feasibility and safety of a new single-port robotic surgical system for gynecological surgery in the porcine model. MATERIALS AND METHODS: Six female Tibetan miniature pigs underwent robot-assisted single-port laparoscopic total hysterectomy with the newly developed single-port EDGE SP1000 platform. Estimated blood loss (EBL), docking time, operative time, and intraoperative and postoperative complications were recorded. Postoperative pain was assessed by VAS (visual analog scale) score at 6 h, 12 h, 24 h, and 48 h. Then the experimental animals were observed for one week after surgery to assess their mental status, incisional infections and finally euthanized for necropsy to assess the recovery of the vaginal stump. RESULTS: Six hysterectomies of pigs were successfully completed. There were no significant intraoperative complications in the six surgeries. The average total operation time was 113.33 min (95-143 min), and the average docking time was 5.5 min (4-7 min). The average EBL was 10 ml (5 ~ 20 ml). The mean VAS scores at 6, 12, 24, and 48 h postoperatively were 6.3, 5.7, 5, 3, respectively. At 7 days postoperatively, no significant incisional infections or other complications were observed. Post-euthanasia examination of the pelvis showed no significant abnormalities in the vaginal stump. CONCLUSION: This preclinical study of a new single-port surgical system for gynecologic procedures demonstrated the safety and feasibility of the EDGE SP1000 system in porcine models. Further studies are required to assess its clinical utility in the future.

Effect of collagen membrane and of bone substitute on lateral bone augmentation with titanium mesh: An experimental in vivo study.

AIM: The aim of this study was to identify the additional effects of collagen membrane (CM) and of synthetic bone substitute (BS) on lateral bone augmentation of chronic peri-implant defect with titanium mesh (TM). MATERIALS AND METHODS: Atrophic alveolar ridge was induced in six canine mandibles, and 5 peri-implant defects were achieved in each hemi-mandible. Bone augmentation was attempted using the following randomly allocated modalities: (1) Control: no treatment, (2) TM only group: blood clot covered by TM, (3) TM+BS group: BS covered by TM, (4) TM+CM group: blood clot covered by TM and CM, and (5) TM+BS+CM group: BS covered by TM and CM. After 16 weeks of submerged healing, micro-CT and histomorphometric analyses were performed. RESULTS: TM exposure occurred in one case in the TM only group, one case in the TM+CM group, and two cases in the TM+BS+CM group. Histologically, pseudo-periosteum was observed along the inner and outer surfaces of TM, and the directions of the collagen fiber within the pseudo-periosteum differed according to the additional use of CM. In general, the TM only group rendered higher values in vertical defect fill and dimension of the augmented hard tissue in comparison with the other treatment groups. CONCLUSIONS: Due to the small sample size, this pilot study remains inconclusive. Within the limitations of the study, the use of CM and/or BS did not appear to have an additional benefit on lateral bone augmentation of peri-implant defect with TM.

Effects of masticatory loading on bone remodeling around teeth versus implants: Insights from a preclinical model.

OBJECTIVES: Teeth connect to bone via a periodontal ligament, whereas implants connect to bone directly. Consequently, masticatory loads are distributed differently to periodontal versus peri-implant bone. Our objective was to determine how masticatory loading of an implant versus a tooth affected peri-implant versus periodontal bone remodeling. Our hypothesis was that strains produced by functional loading of an implant would be elevated compared with the strains around teeth, and that this would stimulate a greater degree of bone turnover around implants versus in periodontal bone. MATERIALS AND METHODS: Sixty skeletally mature mice were divided into two groups. In the implant group, maxillary first molars (mxM1) were extracted, and after socket healing, titanium alloy implants were positioned subocclusally. After osseointegration, implants were exposed, resin crowns were placed, and masticatory loading was initiated. In the control group, the dentition was left intact. Responses of peri-implant and periodontal bone were measured using micro-CT, histology, bone remodeling assays, and quantitative histomorphometry while bone strains were estimated using finite element (FE) analyses. CONCLUSIONS: When a submerged osseointegrated implant is exposed to masticatory forces, peri-implant strains are elevated, and peri-implant bone undergoes significant remodeling that culminates in new bone accrual. The accumulation of new bone functions to reduce both peri-implant strains and bone remodeling activities, equivalent to those observed around the intact dentition.

Effects of an intensive experimental protocol on health, fertility, and production in transition dairy cows.

Animal experimentation is required to investigate complex physiological relationships and facilitates development of evidence-based knowledge. However, experimental protocols can interfere with the daily routine of the animals, result in stress and pain, and have adverse effects on health and production. The goal of this study was to investigate the effects of an intensive experimental protocol on health traits and production in transition dairy cows. Eighty experimental dairy Holstein cows (EXP group) underwent serial protocol-based clinical and ultrasonographic examinations, puncture of the jugular vein for blood collection or drug application, and liver biopsy samples, 14 d before until 42 d after parturition. Controls (CTR group) included 206 cows from the same herd, which fulfilled the same inclusion criteria and were kept under the same production management but were not handled for the purpose of this study. Procedure-related effects with a potentially negative effect on health and production were recorded. Furthermore, production, fertility and culling traits of the 2 groups (CTR, EXP) were compared. Most procedure-related adverse effects were associated with transcutaneous liver biopsies and included diffuse inflammation of the skin incision in 11.9% (42 of 320), abscessation of the skin or subcutis in 4.6% (11 of 240), and increased liver echogenicity of the biopsy site in 10.4% (27 of 240). The experimental procedures had a negative effect on milk yield at the start [days in milk (DIM) 5-50, difference: 2.3 kg, standard error (SE): 0.8 kg] and end of lactation (DIM 251-300, difference: 2.0 kg, SE: 1.0 kg; DIM 301-350, difference: 2.3 kg, SE: 1.2 kg) resulting in a lower 305-d milk yield in the EXP group than in the CTR group (difference: 472 kg, SE 214 kg). The milk fat % was higher in the EXP group than in the CTR group from 251 DIM onward (DIM 251-300, difference: 0.20%, SE: 0.09%; DIM 301-350, difference: 0.41%, SE: 0.17%). Also, the somatic cell score was higher in the EXP group than in the CTR group, during early (5-50 DIM, difference: 0.43, SE: 0.22) and from mid-lactation onward (DIM 151-200, difference: 0.43, SE: 0.2; DIM 201-250, difference: 0.49, SE: 0.22; DIM 251-300, difference: 0.55, SE: 0.25; DIM 301-350, difference: 0.61, SE: 0.28). Experimental procedures had no effect on first service conception rate and time to pregnancy, but had a positive effect on stillbirth rate with fewer stillbirths in the CTR group (0%) than in the EXP group (3.9%). Furthermore, experimental handling had no effect on time to culling or type of culling, whereby poor production was a more frequent reason noted for culling in the EXP group. Procedure-associated impairment of production in dairy cows is rarely reported and allows the estimation of the effects of such a study protocol on animal health and production. As a limitation for the interpretation of the results, the number of animals included and conduction in one single herd have to be considered.

Cell culture-based production and in vivo characterization of purely clonal defective interfering influenza virus particles.

BACKGROUND: Infections with influenza A virus (IAV) cause high morbidity and mortality in humans. Additional to vaccination, antiviral drugs are a treatment option. Besides FDA-approved drugs such as oseltamivir or zanamivir, virus-derived defective interfering (DI) particles (DIPs) are considered promising new agents. IAV DIPs typically contain a large internal deletion in one of their eight genomic viral RNA (vRNA) segments. Consequently, DIPs miss the genetic information necessary for replication and can usually only propagate by co-infection with infectious standard virus (STV), compensating for their defect. In such a co-infection scenario, DIPs interfere with and suppress STV replication, which constitutes their antiviral potential. RESULTS: In the present study, we generated a genetically engineered MDCK suspension cell line for production of a purely clonal DIP preparation that has a large deletion in its segment 1 (DI244) and is not contaminated with infectious STV as egg-derived material. First, the impact of the multiplicity of DIP (MODIP) per cell on DI244 yield was investigated in batch cultivations in shake flasks. Here, the highest interfering efficacy was observed for material produced at a MODIP of 1E-2 using an in vitro interference assay. Results of RT-PCR suggested that DI244 material produced was hardly contaminated with other defective particles. Next, the process was successfully transferred to a stirred tank bioreactor (500 mL working volume) with a yield of 6.0E+8 PFU/mL determined in genetically modified adherent MDCK cells. The produced material was purified and concentrated about 40-fold by membrane-based steric exclusion chromatography (SXC). The DI244 yield was 92.3% with a host cell DNA clearance of 97.1% (99.95% with nuclease digestion prior to SXC) and a total protein reduction of 97.2%. Finally, the DIP material was tested in animal experiments in D2(B6).A2G-Mx1r/r mice. Mice infected with a lethal dose of IAV and treated with DIP material showed a reduced body weight loss and all animals survived. CONCLUSION: In summary, experiments not only demonstrated that purely clonal influenza virus DIP preparations can be obtained with high titers from animal cell cultures but confirmed the potential of cell culture-derived DIPs as an antiviral agent.

The Capability Maturity Model as a Measure of Culture of Care in Laboratory Animal Science.

Culture of care in Laboratory Animal Science (LAS) refers to a commitment toward improving animal welfare, scientific quality, staff wellbeing, and transparency for all stakeholders, ensuring that the animals and personnel involved are treated with compassion and respect. A strong culture of care can be established by the proactive implementation of the Three Rs, sharing best practices, caring for and respecting animals and colleagues, empowering staff, taking responsibility for our actions, and having a caring leadership. Culture of care, when established, should be evaluated continuously, in order to foster its progress and persistence. Even though several tools for assessing the culture of care within an institution have been proposed, an ultimate standard for measuring the concept is lacking. Here, we review the culture of care concept and propose the 'Capability Maturity Model' as a means of quantifying culture of care in the laboratory animal setting.

Alternatives to Laboratory Animals: Trends in Replacement and the Three Rs.

The significance of contributions in volumes 11-46 (1983 to 2018) of Alternatives to Laboratory Animals in relation to the reduction, refinement and replacement of animal experimentation in biomedical research and testing is reviewed and discussed by the journal's former editor-in-chief, with particular emphasis on the development and production of the journal itself, FRAME, the European Centre for the Validation of Alternative Methods and other organisations. The role of the journal in promoting the principles of humane research (as spelled out by William Russell and Rex Burch) and highlighting a range of important issues and focus topics is explored. These include: botulinum toxin potency testing; ethical issues; the use of human volunteers, and human cells and tissues; the use of non-human primates (especially chimpanzees) and dogs as laboratory animals; toxicity testing in relation to cosmetics, pharmaceuticals and chemicals; UK and EU politics and legislation; and test validation and invalidation. The review concludes by identifying some of the issues that still need to be discussed and some of the questions that urgently need to be addressed.

Dental Pulp Stem Cell Therapy in Ischemic Stroke: A Meta-Analysis of Preclinical Studies.

OBJECTIVE: More preclinical research evidence has shown that dental pulp stem cells (DPSCs) transplantation is expected to promote the recovery of ischemic stroke (IS), but it still lacks an evidence-based analysis. The purpose of this study was to investigate the effects of DPSCs on neurological function and infarct size in Sprague-Dawley (SD) rats with middle cerebral artery embolization (MCAO). METHODS: According to PRISMA guidelines, the preclinical study of DPSCs in the treatment of IS was screened according to the inclusion and exclusion criteria, and the relevant data and quality were evaluated by two independent researchers; A meta-analysis of histological and behavioral results was performed. RESULTS: Seven studies were finally included, with quality evaluation scores ranging from 8 to 9. Four articles reported modified Neurological Severity Scores (mNSS), three studies reported rotarod test, and six studies reported infarct volume. Meta-analysis showed that the mNSS score decreased by 1.17 times, the rotarod test increased by 1.11 times and the volume of cerebral infarction decreased by 1.91 times in the DPSC group compared with the blank control group. CONCLUSION: Transplantation of DPSCs can significantly improve the neurological function of ischemic stroke and reduce the infarct volume.