Biallelic variants in RBM42 cause a multisystem disorder with neurological, facial, cardiac, and musculoskeletal involvement

Here, we report a previously unrecognized syndromic neurodevelopmental disorder associated with biallelic loss-of-function variants in the RBM42 gene. The patient is a 2-year-old female with severe central nervous system (CNS) abnormalities, hypotonia, hearing loss, congenital heart defects, and dysmorphic facial features. Familial whole-exome sequencing (WES) reveals that the patient has two compound heterozygous variants, c.304C>T (p.R102*) and c.1312G>A (p.A438T), in the RBM42 gene which encodes an integral component of splicing complex in the RNA-binding motif protein family. The p.A438T variant is in the RRM domain which impairs RBM42 protein stability in vivo. Additionally, p.A438T disrupts the interaction of RBM42 with hnRNP K, which is the causative gene for Au-Kline syndrome with overlapping disease characteristics seen in the index patient. The human R102* or A438T mutant protein failed to fully rescue the growth defects of RBM42 ortholog knockout ΔFgRbp1 in Fusarium while it was rescued by the wild-type (WT) human RBM42. A mouse model carrying Rbm42 compound heterozygous variants, c.280C>T (p.Q94*) and c.1306_1308delinsACA (p.A436T), demonstrated gross fetal developmental defects and most of the double mutant animals died by E13.5. RNA-seq data confirmed that Rbm42 was involved in neurological and myocardial functions with an essential role in alternative splicing (AS). Overall, we present clinical, genetic, and functional data to demonstrate that defects in RBM42 constitute the underlying etiology of a new neurodevelopmental disease which links the dysregulation of global AS to abnormal embryonic development.

RBM46 is essential for gametogenesis and functions in post-transcriptional roles affecting meiotic cohesin subunits

RBM46 is a germ cell-specific RNA-binding protein required for gametogenesis, but the targets and molecular functions of RBM46 remain unknown. Here, we demonstrate that RBM46 binds at specific motifs in the 3'UTRs of mRNAs encoding multiple meiotic cohesin subunits and show that RBM46 is required for normal synaptonemal complex formation during meiosis initiation. Using a recently reported, high-resolution technique known as LACE-seq and working with low-input cells, we profiled the targets of RBM46 at single-nucleotide resolution in leptotene and zygotene stage gametes. We found that RBM46 preferentially binds target mRNAs containing GCCUAU/GUUCGA motifs in their 3'UTRs regions. In Rbm46 knockout mice, the RBM46-target cohesin subunits displayed unaltered mRNA levels but had reduced translation, resulting in the failed assembly of axial elements, synapsis disruption, and meiotic arrest. Our study thus provides mechanistic insights into the molecular functions of RBM46 in gametogenesis and illustrates the power of LACE-seq for investigations of RNA-binding protein functions when working with low-abundance input materials.

Research progress on the expression of the RBM20 gene in dilated cardiomyopathy / 中国当代儿科杂志

Dilated cardiomyopathy (DCM) is a significant contributor to heart failure and can lead to life-threatening cardiovascular events at any stage. RNA-binding motif protein 20 (RBM20) gene mutation is known to be one of the causes of DCM. This mutation exhibits familial aggregation and is associated with arrhythmias, increasing the risk of sudden and early death. This article delves into the characteristics of the RBM20 gene, highlighting its role in regulating alternative splicing of the TTN gene and calcium/calmodulin-dependent protein kinase type II gene. Furthermore, the article provides a summary of treatment options available for DCM caused by RBM20 gene mutations, aiming to enhance clinicians' understanding of the RBM20 gene and provide new ideas for precision medicine treatment.

Effect and Mechanism of RBM8A on Proliferation, Migration and Apoptosis of Human Endometrial Cancer HEC-1A Cells / 肿瘤防治研究

Objective To investigate the effect of silenced RBM8A gene on the biological behavior (proliferation, migration, and apoptosis) of human endometrial cancer HEC-1A cells and its possible mechanism. Methods The hairpin shRNA targeted by the RBM8A gene was designed, and the best shRNA silencing fragment was screened. The recombinant lentiviral interference vector carrying the target gene was constructed and used to infect HEC-1A cells. Cells with stable knockdown of RBM8A gene were screened by puromycin as the experimental group (shRBM8A), while the shRNA of nonsense sequence was designed as the control group (shControl). CCK-8 method was used to detect cell proliferation, and flow cytometry was used to detect cell apoptosis. Transwell assay was used to detect cell migration and invasion. Western blot was used to analyze the expression of apoptosis-related proteins and EMT signal transduction pathway related proteins. Results In comparison with the shControl group, after RBM8A knockdown, HEC-1A cell proliferation was reduced, apoptosis was increased, migration and invasion ability were significantly inhibited (P < 0.05), the expression of apoptosis-related proteins cleaved caspase 9 and caspase 3 increased, EMT-related protein E-cadherin expression increased, and Vimentin expression decreased. Conclusion RBM8A gene silencing can inhibit the proliferation, migration, and invasion and promote the apoptosis of endometrial cancer cells. The inhibition of EMT signal transduction pathway may be its mechanism.

Mild hypothermia alleviates renal ischemia-reperfusion injury by up-regulating the expression of cold-shock protein RBM3 / 器官移植

Objective To evaluate the effect of mild hypothermia on the renal ischemia-reperfusion injury (IRI), and the expression profile of RNA-binding motif protein 3(RBM3) and its downstream effector molecules during this process. Methods Eighteen healthy SD male rats were randomly divided into the normal control (NC) group, IRI group and mild hypothermia pretreat (MHP) group, with 6 rats in each group. Serum creatinine level was measured to evaluate the renal function. Hematoxylin-eosin (HE) staining was performed to assess the renal tissue injury. Western blot was used to determine the relative expression levels of RBM3, Yes-associated protein 1(YAP1), nuclear factor E2-related factor 2(Nrf2), B cell-lymphoma-2(Bcl-2) and Bcl-2-associated X protein (Bax) in the kidney tissues. Immunohistochemical staining was employed to further detect the expression levels of RBM3 and YAP1 proteins. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was adopted to detect the cell apoptosis of kidney tissues. Malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were evaluated to determine the oxidative stress level of kidney tissues. Results Compared with the NC group, the serum creatinine level, the pathological injury score of kidney tissues and the expression levels of RBM3, YAP1 and Nrf2 proteins were significantly up-regulated, the Bcl-2/Bax ratio was considerably lower, the apoptosis rate was remarkably elevated, the MDA content was significantly increased and the SOD activity was dramatically reduced in the IRI and MHP groups (all P < 0.05). Compared with the IRI group, the serum creatinine level and the pathological injury score of kidney tissues were significantly decreased, the expression levels of RBM3, YAP1 and Nrf2 proteins were significantly up-regulated, the Bcl-2/Bax ratio was considerably higher, the apoptosis rate was significantly decreased, the MDA content was significantly decreased and the SOD activity was considerably elevated in the MHP group (all P < 0.05). Conclusions Mild hypothermia may exert protective effect upon renal IRI and it could alleviate cell apoptosis and oxidative stress injury induced by IRI, probably by up-regulating the expression level of RBM3 and its downstream effector molecules of YAP1 and Nrf2.

Prognostic value of increased expression of RBM8A in gastric cancer

This study was designed to investigate the expression of RBM8A protein in patients with gastric cancer (GC) and to explore its correlation with clinical pathological features as well as prognosis. One hundred pairs of gastric carcinoma tissues and adjacent tissues from patients undergoing gastrectomy for GC were included in this study. The protein expression level of RBM8A was determined by immunohistochemistry using tissue microarrays. We also detected the mRNA expression level of RBM8A in 16 pairs of gastric carcinoma tissues and adjacent tissues. Meanwhile, we predicted the potential correlation between RBM8A and tumor stages as well as survival condition in patents with GC based on The Cancer Genome Atlas (TCGA) database. The correlation of RBM8A with the clinical pathological features and prognosis of the 100 patients with GC was also elucidated. The expression level of RBM8A was significantly higher in gastric carcinoma tissues compared to the adjacent tissues. The protein level of RBM8A was correlated with tumor size (P=0.031), depth of invasion (P<0.001), lymph node metastasis (P<0.001), TNM stage (<0.001), and distant metastasis (P=0.001). Patients with increased RBM8A expression (P<0.0018, 95%CI=0.322−0.871), higher TNM stage (P<0.001, 95%CI=4.990−11.283), and lymph node metastasis (P<0.001, 95%CI=2.873−4.002) had a lower overall survival. Taken together, our study demonstrated that RBM8A may act as a proto-oncogene, which could be a promising biomarker and therapeutic target in the diagnosis and treatment of GC.

RNA binding protein 24 deletion disrupts global alternative splicing and causes dilated cardiomyopathy

RNA splicing contributes to a broad spectrum of post-transcriptional gene regulation during normal development, as well as pathological manifestation of heart diseases. However, the functional role and regulation of splicing in heart failure remain poorly understood. RNA binding protein (RBP), a major component of the splicing machinery, is a critical factor in this process. RNA binding motif protein 24 (RBM24) is a tissue-specific RBP which is highly expressed in human and mouse heart. Previous studies demonstrated the functional role of RBM24 in the embryonic heart development. However, the role of RBM24 in postnatal heart development and heart disease has not been investigated. In this paper, using conditional RBM24 knockout mice, we demonstrated that ablation of RBM24 in postnatal heart led to rapidly progressive dilated cardiomyopathy (DCM), heart failure, and postnatal lethality. Global splicing profiling revealed that RBM24 regulated a network of genes related to cardiac function and diseases. Knockout of RBM24 resulted in misregulation of these splicing transitions which contributed to the subsequent development of cardiomyopathy. Notably, our analysis identified RBM24 as a splice factor that determined the splicing switch of a subset of genes in the sacomeric Z-disc complex, including Titin, the major disease gene of DCM and heart failure. Together, this study identifies regulation of RNA splicing by RBM24 as a potent player in remodeling of heart during postnatal development, and provides novel mechanistic insights to the pathogenesis of DCM.

RNA binding protein 24 regulates the translation and replication of hepatitis C virus

The secondary structures of hepatitis C virus (HCV) RNA and the cellular proteins that bind to them are important for modulating both translation and RNA replication. However, the sets of RNA-binding proteins involved in the regulation of HCV translation, replication and encapsidation remain unknown. Here, we identified RNA binding motif protein 24 (RBM24) as a host factor participated in HCV translation and replication. Knockdown of RBM24 reduced HCV propagation in Huh7.5.1 cells. An enhanced translation and delayed RNA synthesis during the early phase of infection was observed in RBM24 silencing cells. However, both overexpression of RBM24 and recombinant human RBM24 protein suppressed HCV IRES-mediated translation. Further analysis revealed that the assembly of the 80S ribosome on the HCV IRES was interrupted by RBM24 protein through binding to the 5'-UTR. RBM24 could also interact with HCV Core and enhance the interaction of Core and 5'-UTR, which suppresses the expression of HCV. Moreover, RBM24 enhanced the interaction between the 5'- and 3'-UTRs in the HCV genome, which probably explained its requirement in HCV genome replication. Therefore, RBM24 is a novel host factor involved in HCV replication and may function at the switch from translation to replication.

miR-483-5p promotes prostate cancer cell proliferation and invasion by targeting RBM5

ABSTRACT Objective: miR-483-5p has been identified as a miRNA oncogene in certain cancers. However, its role in prostate cancer has not been sufficiently investigated. In this study, we investigated the role of miR-483-5p in prostate cancer and examined RBM5 regulation by miR-483-5p. Material and methods: Expression levels of miR-483-5p were determined by quantitative real-time PCR. The effect of miR-483-5p on proliferation was evaluated by MTT assay, cell invasion was evaluated by trans-well invasion assays, and target protein expression was determined by western blotting in LNCaP, DU-145, and PC-3 cells. Luciferase reporter plasmids were constructed to confirm the action of miR-483-5p on downstream target gene RBM5 in HEK-293T cells. Results: we observed that miR-483-5p was upregulated in prostate cancer cell lines and tissues. A miR-483-5p inhibitor inhibited prostate cancer cell growth and invasion in DU-145 and PC-3 cells. miR-483-5p directly bound to the 3' untranslated region (3'UTR) of RBM5 in HEK-293T cells. RBM5 overexpression inhibited prostate cancer cell growth and invasion in LNCaP cells. Enforced RBM5 expression alleviated miR-483-5p promotion of prostate cancer cell growth and invasion in LNCaP cells. Conclusion: The present study describes a potential mechanism underlying a miR-483-5p/RBM5 link that contributes to prostate cancer development.

The effect of blasting and anodizing-combined treatment of implant surface on response of osteoblast-like cell / 대한치과보철학회지

PURPOSE: The purpose of this study is to examine characteristics of implant surface with RBM and anodizing treatments, and to evaluate the responses of osteoblast-like cell (MG-63 cell). MATERIALS AND METHODS: Grade IV titanium disks were fabricated (Diameter 10 mm, thickness 3 mm). Anodizing treatment (ASD) group, RBM and anodizing treatment (RBM/ASD) group, control (machined surface) group were divided. In this study, osteoblast-like cell was used for experiments. The experiments consist of surface characteristics evaluation by FE-SEM images, energy dispersive spectroscopy and stereo-SEM. In order to evaluate cell adhesion evaluation by crystal violet assay and observe cells form by confocal laser microscopy. To assess cell proliferation by XTT assay, cell differentiation by RT-PCR and mineralization by Alizarin red S stain assay. ELISA analyzer was used for Quantitative evaluation. Comparative analysis was run by one-way ANOVA (SPSS version 18.0). Differences were considered statistically significant at P<.05. RESULTS: In ASD group and RBM/ASD group, the surface shape of the crater was observed and components of oxygen and phosphate ions in comparison with the control group were detected. The surface average roughness was obtained 0.08 +/- 0.04 microm in the control group, 0.52 +/- 0.14 microm in ASD group and 1.45 +/- 0.25 microm in RBM/ASD group. In cell response experiments, ASD group and RBM/ASD group were significantly higher values than control group in cell adhesion and mineralization phase, control group was the highest values in the proliferative phase. In RT-PCR experiments, RBM/ASD group was showed higher ALP activity than other groups. RBM/ASD group in comparison with ASD group was significantly higher value for cell adhesion and proliferation phase. CONCLUSION: In the limitation of this study, we are concluded that the surface treatment with RBM/ASD seems more effective than ASD alone or machined surface on cellular response.

Analysis of attachment, proliferation and differentiation response of human mesenchymal stem cell to various implant surfaces coated with rhBMP-2 / 대한치과보철학회지

PURPOSE: In this paper we tried to evaluate the most appropriate surface for rhBMP-2 coating among 4 rough titanium surfaces. MATERIALS AND METHODS: We used machined surface as a control group and anodized, RBM and SLA surfaces as test groups. We coated rhBMP-2 on the 4 surfaces and with uncoated surfaces for each case, we cultured human mesenchymal stem cells on all 8 surfaces. 24 hours after we measured the stem cell'attachment with SEM, and on 3rd, 7th, and 14th days, we checked the cell proliferation and differentiation by using MTT and ALP activity assay. And on the 7th day after the culture, we performed RT-PCR assay to determine whether the expression levels of Type I collagen, osteocalcin, osteopontin were changed. RESULTS: We observed with SEM that 4 rhBMP-2 coated surfaces exhibited wider and tighter cell attachment and more cell process spreading than uncoated surfaces. The anodized rhBMP-2 surface caused robustest effects. In MTT assay we could not find any meaningful difference. In ALP assay there was a significant increase (P<.05) in the ALP activity of anodized rhBMP-2 coated surface compared with that of the control (3rd and 14th days) and with that of the RBM rhBMP-2 coated surface (14th day). In RT-PCR assay there was increased expressions in the anodized rhBMP-2 coated surface for osteocalcin, and osteopontin. CONCLUSION: We found that the anodized rhBMP-2 coated surface were most prominent stem cell attachment and differentiation in compared to control and Machined rhBMP-2 coated, RBM rhBMP-2 coated surface.

Rhipicephalus (Boophilus) microplus: expression and characterization of Bm86-CG in Pichia pastoris / Rhipicephalus (Boophilus) microplus: expressão e caracterização da Bm86-CG em Pichia pastoris

The cattle tick Rhipicephalus (Boophilus) microplus is responsible for great economic losses. It is mainly controlled chemically, with limitations regarding development of resistance to the chemicals. Vaccines may help control this parasite, thereby reducing tick pesticide use. In this light, we performed subcloning of the gene of the protein Bm86-GC, the homologue protein that currently forms the basis of vaccines (GavacTM and TickGardPLUS) that have been developed against cattle ticks. The subcloning was done in the pPIC9 expression vector, for transformation in the yeast Pichia pastoris. This protein was characterized by expression of the recombinant Mut+ strain, which expressed greater quantities of protein. The expressed protein (rBm86-CG) was recognized in the Western-blot assay using anti-Gavac, anti-TickGard, anti-larval extract and anti-rBm86-CG polyclonal sera. The serum produced in cattle vaccinated with the antigen CG rBm86 presented high antibody titers and recognized the native protein. The rBm86-GC has potential relevance as an immunogen for vaccine formulation against cattle ticks.

O carrapato-do-boi Rhipicephalus (Boophilus) microplus é responsável por grandes perdas econômicas. Seu controle é principalmente químico e apresenta limitações quanto ao desenvolvimento de resistência aos princípios ativos. As vacinas podem auxiliar no controle deste parasita diminuindo as aplicações de carrapaticidas. Considerando isso, foi realizada a subclonagem do gene da proteína Bm86-CG, proteína homologa a que atualmente é a base das vacinas desenvolvidas (GavacTM e TickGardPLUS) contra o carrapato-do-boi, no vetor de expressão pPIC9, para ser transformado em levedura, Pichia pastoris. Esta proteína foi caracterizada pela expressão da cepa recombinante Mut+ que expressou maior quantidade de proteína. A proteína expressa, rBm86-CG, foi reconhecida no ensaio de Western-blot pelos soros policlonais anti-Gavac, anti-TickGard, anti-Extrato de larva e anti-rBm86-CG. O soro produzido em bovinos vacinados com o antígeno rBm86-CG apresentou altos títulos de anticorpo e reconheceu a proteína nativa. A rBm86-CG possui potencial relevância como imunógeno para formulação vacinal contra o carrapato de bovinos.

Influence of surface treatment on the insertion pattern of self-drilling orthodontic mini-implants / 대한치과교정학회지

OBJECTIVE: The purpose of this study was to compare self-drilling orthodontic mini-implants of different surfaces, namely, machined (untreated), etched (acid-etched), RBM (treated with resorbable blasting media) and hybrid (RBM + machined), with respect to the following criteria: physical appearance of the surface, measurement of surface roughness, and insertion pattern. METHODS: Self-drilling orthodontic mini-implants (Osstem implant, Seoul, Korea) with the abovementioned surfaces were obtained. Surface roughness was measured by using a scanning electron microscope and surface-roughness-testing machine, and torque patterns and vertical loadings were measured during continuous insertion of mini-implants into artificial bone (polyurethane foam) by using a torque tester of the driving-motor type (speed, 12 rpm). RESULTS: The mini-implants with the RBM, hybrid, and acid-etched surfaces had slightly increased maximum insertion torque at the final stage (p < 0.05). Implants with the RBM surface had the highest vertical load for insertion (p < 0.05). Testing for surface roughness revealed that the implants with the RBM and hybrid surfaces had higher Ra values than the others (p < 0.05). Scanning electron microscopy showed that the implants with the RBM surface had the roughest surface. CONCLUSIONS: Surface-treated, self-drilling orthodontic mini-implants may be clinically acceptable, if controlled appropriately.

Short-Term Retrospective Clinical Study of Resorbable Blasting Media Surface Tapered Implants

Success rate and marginal bone loss of Osstem USII plus implants; Short term clinical study / 대한치과보철학회지

PURPOSE: The aim of this study was to evaluate the clinical value of Osstem(R) USII plus system implants. Clinical and radiographic data were analyzed for 88 implants placed and functionally loaded for a 12 month period at the Yonsei University Dental Hospital. MATERIALS AND METHOD: Based on the patient's medical records, clinical factors and their effects on implant marginal bone resorption, distribution and survival rate were analyzed. The marginal bone loss was evaluated at implant placement and during a 6 to 12 months functional loading period. The independent sample t-test was used to evaluate the interrelationship between the factors (alpha=0.05), and one way repeated measures ANOVA was used to compare the amount of marginal bone resorption. RESULTS: The cumulative survival rate for 88 implants was 100%. The marginal bone resorption from implant placement to prosthetic delivery was 0.24 mm and the average marginal bone resorption from prosthetic delivery to 12 months of functional loading was 0.19 mm. The total average bone resorption from implant placement to 12 months of functional loading was 0.43 mm. There were no statistically differences in the amount of marginal bone resorption when implants were placed in the maxilla or the mandible (P>.05), however, implants placed in the posterior areas showed significantly more marginal bone loss than those placed in the anterior areas (P<.05). CONCLUSION: Based on these results, the short term clinical success rate of RBM surface treated external connection domestic implants showed satisfactory results and the marginal bone loss was in accord with the success criteria of dental implants.

Effect of implant surface microtopography by hydroxyapatite grit-blasting on adhesion, proliferation, and differentiation of osteoblast-like cell line, MG-63

OBJECTIVE: This study examined the potential of the in vitro osteogenesis of microtopographically modified surfaces, RBM (resorbable blasting media) surfaces, which generate hydroxyapatite grit-blasting. METHODS: RBM surfaces were modified hydroxyapatite grit-blasting to produce microtopographically modified surfaces and the surface morphology, roughness or elements were examined. To investigate the potential of the in vitro osteogenesis, the osteoblastic cell adhesion, proliferation, and differentiation were examined using the human osteoblast-like cell line, MG-63 cells. Osteoblastic cell proliferation was examined as a function of time. In addition, osteoblastic cell differentiation was verified using four different methods of an ALP activity assay, a mineralization assay using alizarin red-s staining, and gene expression of osteoblastic differentiation marker using RT-PCR or ELISA. RESULTS: Osteoblastic cell adhesion, proliferation and ALP activity was elevated on the RBM surfaces compared to the machined group. The cells exhibited a high level of gene expression of the osteoblastic differentiation makers (osteonectin, type I collagen, Runx-2, osterix). imilar data was represented in the ELISA produced similar results in that the RBM surface increased the level of osteocalcin, osteopontin, TGF-beta1 and PGE2 secretion, which was known to stimulate the osteogenesis. Moreover, alizarin red-s staining revealed significantly more mineralized nodules on the RBM surfaces than the machined discs. CONCLUSION: RBM surfaces modified with hydroxyapatite grit-blasting stimulate the in vitro osteogenesis of MG-63 cells and may accelerate bone formation and increase bone-implant contact.

Er: YAG Laser Irradiated Implant Surface Observation with Scanning Electron Microscopy

A compare research of machined implant and RBM surface implant / 대한치주과학회지

PURPOSE: To evaluate RBM surface implant which has its good character like that good initial stability, early bone formation. MATERIAL AND METHODS: In this study fixures have divided in 2 group: Machined(Group I), RBM(Group II). Total 10 fixtures were implanted on rabbit which sacrificed on week 2 and week 4 for the histological specimens. By these specimens polarized microscopic view, micro CT view, ISQ value were measured, compared and analysed by each group to figure out the evidence that clinical use of RBM implant. RESULT: ISQ value had no significance differences between 2 groups, However in each group 4, 8 weeks had higher ISQ value than 2 weeks. In polarized microscope, calcification level was following : Group II, Group I. In micro CT, formation of cancellous bone level was following : Group II, Group I. CONCLUSION: RBM implant was the most excellent on the early bone formation and good initial stability.

EPMA analysis of bone formation around RBM surface implant / 대한치주과학회지

PURPOSE: To evaluate bone formation on the implant surface between machined fixture and RBM surface fixture through analyzing of distribution and concentration of Ca and P by EPMA. MATERIAL AND METHODS: Fixtures had divided in 2 group: Machined(Group I), RBM (Group II). Total 4 fixtures were implanted on rabbit which sacrificed on 2 week and 4 week for the histological specimens. By these specimens EPMA value were measured, compared and analysed by each group to figure out the evidence of clinical use of RBM implant. RESULT: After 2 weeks and 4 weeks , it was analyzed that bone formation area, distribution and concentration of Ca and P by EPMA. In distribution and concentration of P Group II was higher than Group I, but there were no statistical significances. In new bone formation area, Group II was more higher than Group I with statistically significances. Both of group, after 4 weeks area is little bit higher than after 2 weeks area but there is no statistically significances. CONCLUSION: RBM implant was better than machined implant on the early bone formation.

Effects of different oxidized surfaces of implant on osseointegration; resonance frequency and histomorphometric analysis study in mini-pigs