High-sensitivity strain sensor based on an asymmetric tapered air microbubble Fabry-Pérot interferometer with an ultrathin wall.

A Fabry-Pérot interferometer (FPI) with an asymmetric tapered structure and air microbubble with an ultrathin wall is designed for high-sensitivity strain measurement. The sensor contains an air microbubble formed by two single-mode fibers (SMF) prepared by fusion splicer arc discharge, and a taper is applied to one side of the air microbubble with a wall thickness of 3.6 µm. In this unique asymmetric structure, the microbubble is more easily deformed under stress, and the strain sensitivity of the sensor is up to 15.89 pm/µÉ› as evidenced by experiments.The temperature sensitivity and cross-sensitivity of the sensor are 1.09 pm/°C and 0.069 µÉ›/°C in the temperature range of 25-200°C, respectively, thus reducing the measurement error arising from temperature variations. The sensor has notable virtues such as high strain sensitivity, low-temperature sensitivity, low-temperature cross-sensitivity, simple and safe process preparation, and low cost. Experiments confirm that the sensor has good stability and repeatability, and it has high commercial potential, especially strain measurements in complex environments.

Multi-wavelength unidirectional forward scattering properties of the arrow-shaped gallium phosphide nanoantenna.

An arrow-shaped gallium phosphide nanoantenna exhibits both near-field electric field enhancement and far-field unidirectional scattering, and the interference conditions involve electric and magnetic quadrupoles as well as toroidal dipoles. By using long-wavelength approximation and exact multipole decomposition, the interference conditions required for far-field unidirectional transverse light scattering and backward near-zero scattering at multiple wavelengths are determined. The near-field properties are excellent, as exemplified by large Purcell factors of 4.5×109 for electric dipole source excitation, 464.68 for magnetic dipole source excitation, and 700 V/m for the field enhancement factor. The degree of enhancement of unidirectional scattering is affected by structural parameters such as the angle and thickness of the nanoantenna. The arrow-shaped nanoantenna is an efficient platform to enhance the electric field and achieve high directionality of light scattering. Moreover, the nanostructure enables flexible manipulation of light waves and materials, giving rise to superior near-field and far-field performances, which are of great importance pertaining to the practicability and application potential of optical antennas in applications such as spectroscopy, sensing, displays, and optoelectronic devices.

Design of optical anapole modes of all-dielectric nanoantennas for SERS applications.

To obtain large electric field enhancement while mitigating material losses, an all-dielectric nanoantenna composed of a heptamer and nanocubes is designed and analyzed. A numerical simulation by the finite element method reveals that the nanoantenna achieves the optical electric anapole modes, thereby significantly enhancing the coupling between different dielectrics to further improve the near-field enhancement and spontaneous radiation. Field enhancement factors |E/E 0|2 of 3,563 and 5,395 (AM1 and AM2) and a Purcell factor of 3,872 are observed in the wavelength range between 350 and 800 nm. This nanoantenna has promising potential in applications involving surface-enhanced Raman scattering and nonlinearities due to its low cost and excellent compatibility.

Analyses of the Cellular Interactions between the Ossification of Collagen-Based Barrier Membranes and the Underlying Bone Defects.

Barrier membranes are an essential tool in guided bone Regeneration (GBR), which have been widely presumed to have a bioactive effect that is beyond their occluding and space maintenance functionalities. A standardized calvaria implantation model was applied for 2, 8, and 16 weeks on Wistar rats to test the interactions between the barrier membrane and the underlying bone defects which were filled with bovine bone substitute materials (BSM). In an effort to understand the barrier membrane's bioactivity, deeper histochemical analyses, as well as the immunohistochemical detection of macrophage subtypes (M1/M2) and vascular endothelial cells, were conducted and combined with histomorphometric and statistical approaches. The native collagen-based membrane was found to have ossified due to its potentially osteoconductive and osteogenic properties, forming a "bony shield" overlying the bone defects. Histomorphometrical evaluation revealed the resorption of the membranes and their substitution with bone matrix. The numbers of both M1- and M2-macrophages were significantly higher within the membrane compartments compared to the underlying bone defects. Thereby, M2-macrophages significantly dominated the tissue reaction within the membrane compartments. Statistically, a correlation between M2-macropahges and bone regeneration was only found at 2 weeks post implantationem, while the pro-inflammatory limb of the immune response correlated with the two processes at 8 weeks. Altogether, this study elaborates on the increasingly described correlations between barrier membranes and the underlying bone regeneration, which sheds a light on the understanding of the immunomodulatory features of biomaterials.

Suitability of R. pulmo Jellyfish-Collagen-Coated Well Plates for Cytocompatibility Analyses of Biomaterials.

Cytocompatibility analyses of new implant materials or biomaterials are not only prescribed by the Medical Device Regulation (MDR), as defined in the DIN ISO Norm 10993-5 and -12, but are also increasingly replacing animal testing. In this context, jellyfish collagen has already been established as an alternative to mammalian collagen in different cell culture conditions, but a lack of knowledge exists about its applicability for cytocompatibility analyses of biomaterials. Thus, the present study was conducted to compare well plates coated with collagen type 0 derived from Rhizostoma pulmo with plates coated with bovine and porcine collagen. The coated well plates were analysed in vitro for their cytocompatibility, according to EN ISO 10993-5/-12, using both L929 fibroblasts and MC3T3 pre-osteoblasts. Thereby, the coated well plates were compared, using established materials as positive controls and a cytotoxic material, RM-A, as a negative control. L929 cells exhibited a significantly higher viability (#### p < 0.0001), proliferation (## p < 0.01), and a lower cytotoxicity (## p < 0.01 and # p < 0.05)) in the Jellagen® group compared to the bovine and porcine collagen groups. MC3T3 cells showed similar viability and acceptable proliferation and cytotoxicity in all collagen groups. The results of the present study revealed that the coating of well plates with collagen Type 0 derived from R. pulmo leads to comparable results to the case of well plates coated with mammalian collagens. Therefore, it is fully suitable for the in vitro analyses of the cytocompatibility of biomaterials or medical devices.

The Use of Collagen-Based Materials in Bone Tissue Engineering.

Synthetic bone substitute materials (BSMs) are becoming the general trend, replacing autologous grafting for bone tissue engineering (BTE) in orthopedic research and clinical practice. As the main component of bone matrix, collagen type I has played a critical role in the construction of ideal synthetic BSMs for decades. Significant strides have been made in the field of collagen research, including the exploration of various collagen types, structures, and sources, the optimization of preparation techniques, modification technologies, and the manufacture of various collagen-based materials. However, the poor mechanical properties, fast degradation, and lack of osteoconductive activity of collagen-based materials caused inefficient bone replacement and limited their translation into clinical reality. In the area of BTE, so far, attempts have focused on the preparation of collagen-based biomimetic BSMs, along with other inorganic materials and bioactive substances. By reviewing the approved products on the market, this manuscript updates the latest applications of collagen-based materials in bone regeneration and highlights the potential for further development in the field of BTE over the next ten years.

Barrier Membranes for Guided Bone Regeneration (GBR): A Focus on Recent Advances in Collagen Membranes.

Guided bone regeneration (GBR) has become a clinically standard modality for the treatment of localized jawbone defects. Barrier membranes play an important role in this process by preventing soft tissue invasion outgoing from the mucosa and creating an underlying space to support bone growth. Different membrane types provide different biological mechanisms due to their different origins, preparation methods and structures. Among them, collagen membranes have attracted great interest due to their excellent biological properties and desired bone regeneration results to non-absorbable membranes even without a second surgery for removal. This work provides a comparative summary of common barrier membranes used in GBR, focusing on recent advances in collagen membranes and their biological mechanisms. In conclusion, the review article highlights the biological and regenerative properties of currently available barrier membranes with a particular focus on bioresorbable collagen-based materials. In addition, the advantages and disadvantages of these biomaterials are highlighted, and possible improvements for future material developments are summarized.

In Vivo Analysis of the Immune Response to Strontium- and Copper-doped Bioglass.

BACKGROUND: Bioglass is a highly adoptable bone substitute material which can be combined with so-called therapeutic ions. However, knowledge is poor regarding the influence of therapeutic ions on immune reactions and associated bone healing. Thus, the aim of this work was to investigate the influence of strontium- and copper-doped bioglass on the induction of M1 and M2 macrophages, as well as vascularization. MATERIALS AND METHODS: Two types of alkali glass were produced based on ICIE16 bioglass via the melt-quench method with the addition of 5 wt% copper or strontium (ICIE16-Cu and ICIE16-Sr). Pure ICIE16 and 45S5 bioglass were used as control materials. The ion release and chemical composition of the bioglass were investigated, and an in vivo experiment was subcutaneously performed on Sprague-Dawley rats. RESULTS: Scanning electron microscopy revealed significant differences in the surface morphology of the bioglass materials. Energy dispersive X-ray spectroscopy confirmed the efficiency of the doping process by showing the ion-release kinetics. ICIE16-Cu exhibited a higher ion release than ICIE16-Sr. ICIE16-Cu induced low immune cell migration and triggered not only a low number of M1 and M2 macrophages but also of blood vessels. ICIE16-Sr induced higher numbers of M1 macrophages after 30 days. Both bioglass types induced numbers of M2 macrophages comparable with those found in the control groups. CONCLUSION: Bioglass doping with copper and strontium did not significantly influence the foreign body response nor vascularization of the implantation bed in vivo. However, all the studied bioglass materials seemed to be biocompatible.

In Vivo Analysis of the Regeneration Capacity and Immune Response to Xenogeneic and Synthetic Bone Substitute Materials.

Although various studies have investigated differences in the tissue reaction pattern to synthetic and xenogeneic bone substitute materials (BSMs), a lack of knowledge exists regarding the classification of both materials based on the DIN ISO 10993-6 scoring system, as well as the histomorphometrical measurement of macrophage subtypes within their implantation beds. Thus, the present study was conducted to analyze in vivo responses to both xenogeneic and synthetic bone substitute granules. A standardized calvaria implantation model in Wistar rats, in combination with established scoring, histological, histopathological, and histomorphometrical methods, was conducted to analyze the influence of both biomaterials on bone regeneration and the immune response. The results showed that the application of the synthetic BSM maxresorb® induced a higher pro-inflammatory tissue response, while the xenogeneic BSM cerabone® induced a higher anti-inflammatory reaction. Additionally, comparable bone regeneration amounts were found in both study groups. Histopathological scoring revealed that the synthetic BSM exhibited non-irritant scores at all timepoints using the xenogeneic BSM as control. Overall, the results demonstrated the biocompatibility of synthetic BSM maxresorb® and support the conclusion that this material class is a suitable alternative to natural BSM, such as the analyzed xenogeneic material cerabone®, for a broad range of indications.

Phosphoproteomics Profile of Chicken Cecum in the Response to Salmonella enterica Serovar Enteritidis Inoculation.

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a foodborne pathogen, which can cause great threats to human health through the consumption of contaminated poultry products. This research combines TMT labeling, HPLC and mass-spectrometry-based phosphoproteomics on cecum of the F1 cross of Guangxi Yao chicken and Jining Bairi chicken. The treated group was inoculated with 0.3 mL inoculum S. Enteritidis, and the control group was inoculated with 0.3 mL phosphate-buffered saline (PBS). A total of 338 differentially phosphorylated modification sites in 243 differentially phosphorylated proteins (DPPs) were chosen for downstream analyses. A total of 213 sites in 146 DPPs were up-regulated and 125 sites in 97 DPPs were down-regulated. Functional analysis was performed for DPPs based on gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and the protein domain. The DPPs were mainly enriched in immune- and metabolic-related GO-BP (biological process) and KEGG pathways. We predicted and classified the subcellular structure and COG/KOG of DPPs. Furthermore, protein-protein interaction network analyses were performed by using multiple algorithms. We identified 71 motifs of the phosphorylated modification sites and selected 18 sites randomly to detect the expression level through parallel reaction monitoring (PRM). S. Enteritidis inoculation caused phosphorylation alteration in immune- and metabolic-related proteins. The invasion of S. Enteritidis may be actualized by inducing cecum cell apoptosis through the endoplasmic reticulum pathway, and chickens could resist the invasion of S. Enteritidis by affecting the function of ECM receptors. The findings herein provide a crucial theoretical foundation to understand the molecular mechanism and epigenetic regulation in response to S. Enteritidis inoculation in chickens.

Mussel-Inspired Carboxymethyl Chitosan Hydrogel Coating of Titanium Alloy with Antibacterial and Bioactive Properties.

Infection-related titanium implant failure rates remain exceedingly high in the clinic. Functional surface coating represents a very promising strategy to improve the antibacterial and bioactive properties of titanium alloy implants. Here, we describe a novel bioactive surface coating that consists of a mussel-inspired carboxymethyl chitosan hydrogel loaded with silver nanoparticles (AgNPs) to enhance the bioactive properties of the titanium alloy. The preparation of hydrogel is based on gallic acid grafted carboxymethyl chitosan (CMCS-GA) catalyzed by DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride). To build a firm bonding between the hydrogel and titanium alloy plate, a polydopamine layer was introduced onto the surface of the titanium alloy. With HRP/H2O2 catalysis, CMCS-GA can simply form a firm gel layer on the titanium alloy plate through the catechol groups. The surface properties of titanium alloy were characterized by scanning electron microscope (SEM), X-ray photoelectron spectroscopy (XPS), and water contact angle. Silver nanoparticles were loaded into the gel layer by in situ reduction to enhance the antibacterial properties. In vitro antibacterial and cell viability experiments showed that the AgNPs-loaded Ti-gel possesses excellent antibacterial properties and did not affect the proliferation of rabbit mesenchymal stem cells (MSCs).

Enhanced Bioavailability of Dihydrotanshinone I-Bovine Serum Albumin Nanoparticles for Stroke Therapy.

Dihydrotanshinone I (DHT) is a natural component in Salvia miltiorrhiza and has been widely researched for its multiple bioactivities. However, poor solubility and biocompatibility of DHT limit its desirable application for clinical purposes. Herein, DHT was encapsulated with bovine serum albumin (BSA) to enhance bioavailability. Compared to free DHT, DHT-BSA NPs (nanoparticles) showed an improved solubility in normal saline and increased protection against hydrogen peroxide-induced oxidative damage in PC12 cells. In addition, DHT-BSA NPs administered by intravenous injection displayed a significant efficacy in the middle cerebral artery occlusion/reperfusion models, without any impact on the cerebral blood flow. In summary, DHT-BSA NPs show an enhanced bioavailability compared with free DHT and a successful penetration into the central nervous system for stroke therapy, demonstrating their application potential in cardio-cerebrovascular diseases.

AtMIF1 increases seed oil content by attenuating GL2 inhibition.

Vegetable oil is a major edible oil and an important industrial raw material. However, breeders have found it challenging to improve the oil content of crop seeds, and little is known about regulators with the potential to increase oil content via molecular engineering in modern oil crop breeding. We reported an F-box protein, Arabidopsis thaliana MYB Interaction Factor 1 (AtMIF1), which is a member of the ubiquitin-protein ligase E3 complex involved in the 26S proteasome protein degradation pathway. AtMIF1 physically interacts with MYB domain protein 5 (MYB5), which results in MYB5 degradation, so that transcriptional activation of the MYB/bHLH/WD-repeat (MBW) complex does not occur normally and GLABRA2 (GL2), encoding an inhibitor of oil content and functioning as a direct downstream gene of MBW, is not properly transcribed. AtMIF1 functioned as a positive regulator that increases oil content by attenuating GL2 inhibition. We overexpressed AtMIF1 and obtained transgenic plants with significantly higher seed oil contents. Importantly, both vegetative and reproductive growth of the transgenic plants appeared normal. In summary, this work reveals a novel regulator, AtMIF1, and a new regulatory pathway, 26S proteasome-AtMIF1-MYB5, for increasing the oil content of seeds without affecting plant growth, thus facilitating oil crop breeding.

Equal parental contribution to the transcriptome is not equal control of embryogenesis.

In animals, early embryogenesis is maternally controlled, whereas in plants, parents contribute equally to the proembryo transcriptome. Thus, the question remains whether equivalent parental contribution to the transcriptome of the early proembryo means equal control of early embryogenesis. Here, on the basis of cell-lineage-specific and allele-specific transcriptome analysis, we reveal that paternal and maternal genomes contribute equally to the transcriptomes of both the apical cell lineage and the basal cell lineage of early proembryos. However, a strong maternal effect on basal cell lineage development was found, indicating that equal parental contribution to the transcriptome is not necessarily coupled with equivalent parental control of proembryonic development. Parental contributions to embryogenesis therefore cannot be concluded solely on the basis of the ratio of paternal/maternal transcripts. Furthermore, we demonstrate that parent-of-origin genes display developmental-stage-dependent and cell-lineage-dependent allelic expression patterns. These findings will facilitate the investigation of specific parental roles in specific processes of early embryogenesis.

Improved aging properties of bio-bitumen coating sheets by using modified lignin.

Papermaking waste liquid (black liquor) is a serious source of water pollution worldwide. The subsequent treatment of it is very difficult cause it contains a large amount of lignin, inorganic salts, organic matter, and pigments, which lead to serious water pollution. Lignin is the main by-product of the paper industry and is the only natural aromatic recyclable resource. Its effective utilization rate is currently less than 3%. Therefore, how to effectively recycle lignin in papermaking waste liquid and further synthesize industrialized products is of great significance to the sustainable development and environmental protection. Besides, based on the shortage of petroleum resources in recent years, the application of biomass resources instead of petroleum resources in the industry is also an important issue. In this article, we explored the best optimal conditions for the oxypropylation and esterification of lignin, and prepared bio-bitumen based on modified lignin, and then applied it to the waterproof coating sheets. FTIR and mechanical properties (softening point, low-temperature flexibility, peel strength, etc.) were tested on the obtained waterproof coating sheets. The results show that the addition of modified lignin reduced the softening point and peel strength of the coating sheets. Interestingly, both oxypropylated lignin (OL) and esterified lignin (OEL) were very beneficial to resist the decrease in peel strength during the aging process, showing a significant improvement in the performance of the coating sheets after aging compared to the control.

Biodegradation of low-density polyethylene by Microbulbifer hydrolyticus IRE-31.

Polyethylene (PE) is one of the most widespread plastic materials. Nevertheless, due to its recalcitrance against biological degradation and the presence of toxic additives, landfilled and carelessly disposed PE products have caused serious pollution in the natural environments. In this work, we aimed to investigate the growth characteristics of Microbulbifer hydrolyticus IRE-31 and its application in the biological degradation of low-density PE. The IRE-31 strain was isolated from marine pulp mill wastes rich in lignin which is a natural complex polymer containing also saturated carbon-carbon bonds like in PE. Following 30 days cultivation of the IRE-31 strain, the biodegradation of linear low-density PE particles was evidenced clearly by morphological changes of the polymer surface monitored by scanning electron microscopy and the formation of additional carbonyl groups in the polymer chains indicated by Fourier transform infrared spectroscopy.

Performance of bitumen coating sheet using biomass pyrolysis oil.

The "green" production of bitumen has raised increasing interest in recent years to reduce the environmental, energy-related and petro-based concerns. Bio-oil, prepared by biomass pyrolysis, can be used as a substitute for petro-based bitumen in bitumen or bitumen-based coatings, for its similar properties of good adhesion and anti-corrosion characteristics as bitumen. Although biomass is a renewable and widespread chemical resource, its high-valued utilization is still difficult. Several studies have qualitatively demonstrated the use of bio-bitumen in practical applications. The present study investigates that adding some bio-oil to traditional bitumen to form a bio-bitumen could help improve the properties of traditional bitumen. Bio-bitumen was prepared from biomass pyrolysis oil and applied to self-adhesive and doped hot-melt sheets. Results of physical properties demonstrate that bio-bitumen is a potential substitute in bitumen coating sheet.Implications: This paper is intended to verify the effect of pyrolyzed bio-oil from wheat straw on the performance of bitumen, as well as the feasibility of application in the coating sheet. Up to now, the research on bio-bitumen is mainly in pavement bitumen. In the present research, bio-bitumen was applied to the coating sheet in different proportions. Interestingly, the prepared coating sheet exhibited higher adhesion. Other performances, such as temperature stability, mechanical strength and temperature flexibility of coating sheet showed improvement in the presence of bio-oil, which indicated the suitability of bio-oil in coating sheet bitumen.

Promoting effect of baicalin on nitric oxide production in CMECs via activating the PI3K-AKT-eNOS pathway attenuates myocardial ischemia-reperfusion injury.

OBJECTIVE: Baicalin, which is isolated from Scutellariae Radix, has been shown to possess therapeutic potential for different diseases. Cardiac microvessel injury in myocardial ischemia-reperfusion (IR) has been extensively explored. However, there have been no studies investigating the physiological regulatory mechanisms of baicalin on nitric oxide production and the necroptosis of cardiac microvascular endothelial cells (CMECs) in myocardial IR injury. This study was designed to investigate the contribution of baicalin to repressing necroptosis and preventing IR-mediated CMEC dysfunction. MATERIALS AND METHODS: Indicators of ventricular structure and function were measured by an echocardiographic system. An MTT assay was performed to assess cell viability. Nitrite detection was performed to detect nitric oxide content, and cGMP content was determined using a commercially available cGMP complete ELISA kit. Morphology and molecular characteristics were detected by electron micrographs, quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. RESULT: Our results demonstrated that baicalin significantly improved cardiac function, decreased the myocardial infarction area, and inhibited myocardial cell apoptosis. Moreover, baicalin had a protective effect on cardiac microvessels and promoted the production of nitric oxide (NO) and the level of cGMP in rats that underwent myocardial IR injury. The results of the in vitro experiments showed that baicalin markedly improved cell activity and function in CMECs exposed to hypoxia-reoxygenation (HR). Further experiments indicated that baicalin supplementation suppressed the protein expression of RIP1, RIP3 and p-MLKL to interrupt CMEC necroptosis. In addition, baicalin promoted the production of NO via activating the PI3K-AKT-eNOS signaling pathway. Taken together, our results identified the PI3K-AKT-eNOS axis as a new pathway responsible for reperfusion-mediated microvascular damage. CONCLUSION: Baicalin protected CMECs in IR rats by promoting the release of NO via the PI3K-AKT-eNOS pathway and mitigated necroptosis by inhibiting the protein expression of RIP1, RIP3 and p-MLKL.

Characterization of single-nucleotide variation in Indian-origin rhesus macaques (Macaca mulatta).

BACKGROUND: Rhesus macaques are the most widely utilized nonhuman primate model in biomedical research. Previous efforts have validated fewer than 900 single nucleotide polymorphisms (SNPs) in this species, which limits opportunities for genetic studies related to health and disease. Extensive information about SNPs and other genetic variation in rhesus macaques would facilitate valuable genetic analyses, as well as provide markers for genome-wide linkage analysis and the genetic management of captive breeding colonies. RESULTS: We used the available rhesus macaque draft genome sequence, new sequence data from unrelated individuals and existing published sequence data to create a genome-wide SNP resource for Indian-origin rhesus monkeys. The original reference animal and two additional Indian-origin individuals were resequenced to low coverage using SOLiD™ sequencing. We then used three strategies to validate SNPs: comparison of potential SNPs found in the same individual using two different sequencing chemistries, and comparison of potential SNPs in different individuals identified with either the same or different sequencing chemistries. Our approach validated approximately 3 million SNPs distributed across the genome. Preliminary analysis of SNP annotations suggests that a substantial number of these macaque SNPs may have functional effects. More than 700 non-synonymous SNPs were scored by Polyphen-2 as either possibly or probably damaging to protein function and these variants now constitute potential models for studying functional genetic variation relevant to human physiology and disease. CONCLUSIONS: Resequencing of a small number of animals identified greater than 3 million SNPs. This provides a significant new information resource for rhesus macaques, an important research animal. The data also suggests that overall genetic variation is high in this species. We identified many potentially damaging non-synonymous coding SNPs, providing new opportunities to identify rhesus models for human disease.

Integrating common and rare genetic variation in diverse human populations.

Despite great progress in identifying genetic variants that influence human disease, most inherited risk remains unexplained. A more complete understanding requires genome-wide studies that fully examine less common alleles in populations with a wide range of ancestry. To inform the design and interpretation of such studies, we genotyped 1.6 million common single nucleotide polymorphisms (SNPs) in 1,184 reference individuals from 11 global populations, and sequenced ten 100-kilobase regions in 692 of these individuals. This integrated data set of common and rare alleles, called 'HapMap 3', includes both SNPs and copy number polymorphisms (CNPs). We characterized population-specific differences among low-frequency variants, measured the improvement in imputation accuracy afforded by the larger reference panel, especially in imputing SNPs with a minor allele frequency of