Different reference genomes determine different results: Comparing SNP calling in RAD-seq of Engelhardia roxburghiana using different reference genomes.

Advances in next-generation sequencing (NGS) have significantly reduced the cost and improved the efficiency of obtaining single nucleotide polymorphism (SNP) markers, particularly through restriction site-associated DNA sequencing (RAD-seq). Meanwhile, the progression in whole genome sequencing has led to the utilization of an increasing number of reference genomes in SNP calling processes. This study utilized RAD-seq data from 242 individuals of Engelhardia roxburghiana, a tropical tree of the walnut family (Juglandaceae), with SNP calling conducted using the STACKS pipeline. We aimed to compare both reference-based approaches, namely, employing a closely related species as the reference genome versus the species itself as the reference genome, to evaluate their respective merits and limitations. Our findings indicate a substantial discrepancy in the number of obtained SNPs between using a closely related species as opposed to the species itself as reference genomes, the former yielded approximately an order of magnitude fewer SNPs compared to the latter. While the missing rate of individuals and sites of the final SNPs obtained in the two scenarios showed no significant difference. The results showed that using the reference genome of the species itself tends to be prioritized in RAD-seq studies. However, if this is unavailable, considering closely related genomes is feasible due to their wide applicability and low missing rate as alternatives. This study contributes to enrich the understanding of the impact of SNP acquisition when utilizing different reference genomes.

Adaptive divergence and genetic vulnerability of relict species under climate change: a case study of Pterocarya macroptera.

BACKGROUND AND AIMS: Understanding adaptive genetic variation and whether it can keep pace with predicted future climate change is critical in assessing the genetic vulnerability of species and developing conservation management strategies. The lack of information on adaptive genetic variation in relict species carrying abundant genetic resources hinders the assessment of genetic vulnerability. Using a landscape genomics approach, this study aimed to determine how adaptive genetic variation shapes population divergence and to predict the adaptive potential of Pterocarya macroptera (a vulnerable relict species in China) under future climate scenarios. METHODS: We applied restriction site-associated DNA sequencing (RAD-seq) to obtain 8244 single-nucleotide polymorphisms (SNPs) from 160 individuals across 28 populations. We examined the pattern of genetic diversity and divergence, and then identified outliers by genetic differentiation (FST) and genotype-environment association (GEA) methods. We further dissected the effect of geographical/environmental gradients on genetic variation. Finally, we predicted genetic vulnerability and adaptive risk under future climate scenarios. KEY RESULTS: We identified three genetic lineages within P. macroptera: the Qinling-Daba-Tianmu Mountains (QDT), Western Sichuan (WS) and Northwest Yunnan (NWY) lineages, which showed significant signals of isolation by distance (IBD) and isolation by environment (IBE). IBD and IBE explained 3.7-5.7 and 8.6-12.8 % of the genetic structure, respectively. The identified GEA SNP-related genes were involved in chemical defence and gene regulation and may exhibit higher genetic variation to adapt to the environment. Gradient forest analysis revealed that the genetic variation was mainly shaped by temperature-related variables, indicating its adaptation to local thermal environments. A limited adaptive potential was suggested by the high levels of genetic vulnerability in marginal populations. CONCLUSIONS: Environmental gradient mainly shaped the population differentiation of P. macroptera. Marginal populations may be at high risk of extinction, and thus proactive management measures, such as assisted gene flow, are required to ensure the survival of these populations.

Complete Chloroplast Genome of an Endangered Species Quercus litseoides, and Its Comparative, Evolutionary, and Phylogenetic Study with Other Quercus Section Cyclobalanopsis Species.

Quercus litseoides, an endangered montane cloud forest species, is endemic to southern China. To understand the genomic features, phylogenetic relationships, and molecular evolution of Q. litseoides, the complete chloroplast (cp) genome was analyzed and compared in Quercus section Cyclobalanopsis. The cp genome of Q. litseoides was 160,782 bp in length, with an overall guanine and cytosine (GC) content of 36.9%. It contained 131 genes, including 86 protein-coding genes, eight ribosomal RNA genes, and 37 transfer RNA genes. A total of 165 simple sequence repeats (SSRs) and 48 long sequence repeats with A/T bias were identified in the Q. litseoides cp genome, which were mainly distributed in the large single copy region (LSC) and intergenic spacer regions. The Q. litseoides cp genome was similar in size, gene composition, and linearity of the structural region to those of Quercus species. The non-coding regions were more divergent than the coding regions, and the LSC region and small single copy region (SSC) were more divergent than the inverted repeat regions (IRs). Among the 13 divergent regions, 11 were in the LSC region, and only two were in the SSC region. Moreover, the coding sequence (CDS) of the six protein-coding genes (rps12, matK, atpF, rpoC2, rpoC1, and ndhK) were subjected to positive selection pressure when pairwise comparison of 16 species of Quercus section Cyclobalanopsis. A close relationship between Q. litseoides and Quercus edithiae was found in the phylogenetic analysis of cp genomes. Our study provided highly effective molecular markers for subsequent phylogenetic analysis, species identification, and biogeographic analysis of Quercus.

An Autograft for Anterior Cruciate Ligament Reconstruction Results in Better Biomechanical Performance and Tendon-Bone Incorporation Than Does a Hybrid Graft in a Rat Model.

BACKGROUND: The biomechanical and tendon-bone incorporation properties of allograft-augmented hybrid grafts for anterior cruciate ligament (ACL) reconstruction compared with traditional autografts are unknown. HYPOTHESIS: Using an autograft for ACL reconstruction yields better results on biomechanical testing, radiographic analysis, and histological evaluation versus using a hybrid graft. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 66 adult male Sprague Dawley rats underwent unilateral ACL reconstruction with an autograft (AT group; n = 33) or a hybrid graft (HB group; n = 33). The grafts used in both groups were harvested from the peroneus longus tendon and were fixed by suturing to the surrounding periosteum. Samples were harvested for biomechanical testing, micro-computed tomography (CT), and histological evaluation at 4, 8, and 12 weeks postoperatively. Bone tunnels on the femoral and tibial sides were divided into 3 subregions: intra-articular (IA), midtunnel (MT), and extra-articular (EA). A cylinder-like volume of interest in the bone tunnel and a tubular-like volume of interest around the bone tunnel were used to evaluate new bone formation and bone remodeling, respectively, via micro-CT. RESULTS: In the AT group, there were significantly higher failure loads and stiffness at 8 weeks (failure load: 3.04 ± 0.40 vs 2.09 ± 0.54 N, respectively; P = .006) (stiffness: 3.43 ± 0.56 vs 1.75 ± 0.52 N/mm, respectively; P < .001) and 12 weeks (failure load: 9.10 ± 1.13 vs 7.14 ± 0.94 N, respectively; P = .008) (stiffness: 4.45 ± 0.75 vs 3.36 ± 0.29 N/mm, respectively; P = .008) than in the HB group. With regard to new bone formation in the bone tunnel, in the AT group, the bone volume/total volume (BV/TV) was significantly higher than in the HB group on the tibial side at 8 weeks (IA: 22.21 ± 4.98 vs 5.16 ± 3.98, respectively; P < .001) (EA: 19.66 ± 7.19 vs 10.85 ± 2.16, respectively; P = .030) and 12 weeks (IA: 30.50 ± 5.04 vs 17.11 ± 7.31, respectively; P = .010) (MT: 21.15 ± 2.58 vs 15.55 ± 4.48, respectively; P = .041) (EA: 20.75 ± 3.87 vs 10.64 ± 3.94, respectively; P = .003). With regard to bone remodeling around the tunnel, the BV/TV was also significantly higher on the tibial side at 8 weeks (MT: 33.17 ± 8.05 vs 15.21 ± 7.60, respectively; P = .007) (EA: 25.19 ± 6.38 vs 13.94 ± 7.10, respectively; P = .030) and 12 weeks (IA: 69.46 ± 4.45 vs 47.80 ± 6.16, respectively; P < .001) (MT: 33.15 ± 3.88 vs 13.76 ± 4.07, respectively; P < .001) in the AT group than in the HB group. Sharpey-like fibers had formed at 8 weeks in the AT group. A large number of fibroblasts withdrew at 12 weeks. In the AT group, the width of the interface was significantly narrower at 4 weeks (85.86 ± 17.49 vs 182.97 ± 14.35 µm, respectively; P < .001), 8 weeks (58.86 ± 10.99 vs 90.15 ± 11.53 µm, respectively; P = .002), and 12 weeks (42.70 ± 7.96 vs 67.29 ± 6.55 µm, respectively; P = .001) than in the HB group. CONCLUSION: Using an autograft for ACL reconstruction may result in improved biomechanical properties and tendon-bone incorporation compared with a hybrid graft. CLINICAL RELEVANCE: Augmenting small autografts with allograft tissue may result in decreased biomechanical performance and worse tendon-bone incorporation, increasing the risk of graft failure.

Tanshinone IIA regulates human AML cell proliferation, cell cycle, and apoptosis through miR-497-5p/AKT3 axis.

BACKGROUND: The roots of Salvia miltiorrhiza are used in traditional Chinese medicine (TCM) and have high medicinal value. Tanshinone IIA (Tan IIA) is the active ingredient of Salvia miltiorrhiza which can inhibit the growth of acute leukemia cell lines in vitro, although the mechanism remains unclear. METHODS: CCK-8 assays and BrdU stain were used to evaluate cell proliferation ability. Western blot analysis was used to detect protein expression. miR-497-5p expression level was detected by using qRT-PCR, and Annexin V-FITC/propidium iodide (PI) was used to detect cell apoptosis. RESULTS: Here we reported that Tan IIA could inhibit cell proliferation, induce cell cycle arrest, and promote cell apoptosis in acute myeloid leukemia (AML) cells. Thus, Tan IIA had the anti-cancer activity in AML cell lines, which was likely mediated by up-regulation of miR-497-5p expression. Our data further showed that in AML cells, the same effects were observed with overexpression of miR-497-5p by a miR-497-5p mimic. We demonstrated that Tan IIA could inhibit the expression of AKT3 by up-regulating the expression of miR-497-5p. We subsequently identified that AKT3 was the direct target of miR-497-5p, and that treatment with Tan IIA obviously reversed the effect of treatment with an miR-497-5p inhibitor under harsh conditions. In turn, PCNA expression was increased and cleaved Caspase-3 was suppressed, which contributed to the growth of AML cells. CONCLUSIONS: Our results showed that Tan IIA could inhibit cell proliferation in AML cells through miR-497-5p-mediated AKT3 downregulation pathway.

Tanshinone â ¡A inhibits VSMC inflammation and proliferation in vivo and in vitro by downregulating miR-712-5p expression.

The inflammation and proliferation of vascular smooth muscle cells (VSMCs) are the basic pathological feature of proliferative vascular diseases. Tanshinone ⅡA (Tan ⅡA), which is the most abundant fat-soluble element extracted from Salvia miltiorrhiza, has potent protective effects on the cardiovascular system. However, the underlying mechanism is still not fully understood. Here, we show that Tan ⅡA significantly inhibits neointimal formation and decreases VSMC inflammation by upregulating the expression of KLF4 and inhibiting the activation of NFκB signaling. Using a microRNA array analysis, we found that miR-712-5p expression is significantly upregulated in tumor necrosis factor alpha (TNF-α)-treated VSMCs. Loss- and gain-of-function experiments revealed that transfection of miR-712-5p mimic promotes, whereas depletion of miR-712-5p suppresses TNF-α-induced VSMC inflammation, leading to amelioration of intimal hyperplasia induced by carotid artery ligation. Moreover, depletion of miR-712-5p by its antagomir largely abrogates TNF-α-induced VSMC proliferation. Our findings suggest that miR-712-5p mediates the stimulatory effect of TNF-α on VSMC inflammation, and that Tan ⅡA inhibits VSMC inflammation and proliferation in vivo and in vitro by suppression of miR-712-5p expression. Targeting miR-712-5p may be a novel therapeutic strategy to prevent proliferative vascular diseases.

Proximal fibular osteotomy alleviates medial compartment knee osteoarthritis in a mouse model.

PURPOSE: The purpose of this study was to establish a mouse model of proximal fibular osteotomy (PFO), and to determine if PFO could delay degeneration of the medial compartment of the knee joint in a mouse model. METHODS: An animal model of destabilization of the medial meniscus (DMM) was used to induce post-traumatic knee osteoarthritis (OA). PFO was performed to examine the effectiveness of PFO on protection against medial compartment knee OA. Micro-CT was used to observe osteosclerosis development in the subchondral bone, and Safranin O-fast green staining was used to evaluate the progression of articular cartilage destruction. The condylar-plateau angle (CPA) and anatomical femorotibial angle (aFTA) were measured to determine whether knee alignment was changed after PFO. RESULTS: PFO treatment could decrease osteophyte formation and osteosclerosis development in the subchondral bone, as observed by micro-CT. The value of the ratio of trabecular bone volume to total volume (BV/TV) of DMM+PFO group was lower than that of DMM group. PFO also inhibited the progression of articular cartilage destruction. DMM + PFO group displayed decreased maximal and summed OA scores, as compared with DMM group. Moreover, the change of knee alignment was reduced by PFO, which might be the mechanism of PFO alleviating medial compartment knee OA. CONCLUSION: Our results indicated that PFO could alleviate medial compartment knee OA in a mouse model.

Synthesis, crystal structures and insulin-like activity of three new oxidovanadium(V) complexes with aroylhydrazone ligand.

Three new oxidovanadium(V) complexes were designed, synthesized and characterized by C, H, N elemental analysis, single crystal X-ray diffraction，UV/Vis and IR spectra. Complex 1: [VOL1X] (H2L1 = (E)-N'-(2-hydroxybenzylidene)-3-methbenzohydrazide, HX = ethylmaltol = 2-ethyl-3-hydroxy-4-pyrone), Complex 2: [VOL2(CH3O)(CH3OH)], (H2L2 = C16H16N2O4 = (E)-N'-(2-hydroxybenzylidene)-3,5-dimethoxybenzohydrazide, CH3OH = methanol), Complex 3: [VOL3X] (H2L3 = (E)-N'-(3-ethoxy-2-hydroxybenzylidene)-3,5-dimethoxybenzohydrazide). The insulin-like activity of the three complexes was tested. Both normal and streptozotocin (STZ)-diabetic mice were administered intragastrically for two weeks. It was found that the complexes at doses of 10.0 and 5.0 mg V·kg-1 can significantly decrease the blood glucose level in STZ-diabetic mice, and the blood glucose level in the treated normal mice was not altered. The lesions of kidney and liver caused by diabetes have varying degrees of improvement.

Comparison of clinical outcomes after anterior cruciate ligament reconstruction with hamstring tendon autograft versus soft-tissue allograft: A meta-analysis of randomised controlled trials.

BACKGROUND: Hamstring tendon autografts and soft-tissue allograft are commonly used for anterior cruciate ligament (ACL) reconstruction. However, the clinical outcomes between these two grafts are controversial. This meta-analysis was performed to compare clinical outcomes of primary ACL reconstruction with hamstring tendon autografts versus soft-tissue allografts. MATERIALS AND METHODS: PubMed, Embase, and the Cochrane Library were searched through 8 September 2017 to identify randomized controlled studies that compared hamstring tendon autografts with soft-tissue allografts for primary ACL reconstruction. Two authors independently graded the methodological quality of each eligible study using the Cochrane Collaboration tool and extracted relevant data. Statistical heterogeneity among the trials was evaluated with chi-square and I-square tests. A sensitivity analysis was conducted to explore sources of heterogeneity. Subgroup analysis was performed to identify potential differences according to type of reconstruction technique (single-bundle or double-bundle). RESULTS: Eight studies with 785 combined patients (396 hamstring tendon autografts and 389 soft-tissue allografts) were included. Two studies had a high risk of bias. The other six studies had unclear risk of bias. There were significant differences between the groups in subjective International Knee Documentation Committee (IKDC) score (mean difference [MD], 2.43; 95%CI, 0.69-4.18; p = 0.006), Tegner score (MD, 0.24; 95%CI, 0.03-0.45; p = 0.03), and side-to-side difference (MD, -1.37; 95%CI, -2.44 to -0.30; p = 0.01). There was no significant difference between the groups in Lysholm score, complications, pivot shift test, anterior drawer test, Lachman test, overall IKDC score, or range of motion. Subgroup analysis demonstrated that for primary ACL reconstruction using the single-bundle technique, soft-tissue allografts were inferior to hamstring tendon autografts in subjective IKDC score, anterior drawer test, and side-to-side difference. CONCLUSION: Soft-tissue allografts are inferior to hamstring tendon autografts with respect to subjective patient evaluation and knee stability but superior in the complication of hypoesthesia for patients undergoing primary ACL reconstruction.

Irradiated allograft versus autograft for anterior cruciate ligament reconstruction: A meta-analysis and systematic review of prospective studies.

BACKGROUND: Irradiated allografts and autografts are commonly used for anterior cruciate ligament (ACL) reconstruction. The outcomes between these two grafts are controversial. This meta-analysis and systematic review of prospective comparative studies was performed to compare the clinical outcomes, including knee functionality, stability, subjective evaluation, complications, and failure, of irradiated allografts and autografts in primary ACL reconstruction. MATERIALS AND METHODS: PubMed, Embase, and the Cochrane Library were searched from database inception to 12 August 2017 to identify prospective studies that compared irradiated allografts with autografts for primary ACL reconstruction. Randomized controlled trials were included in the meta-analysis. Prospective cohort studies were included in the systematic reviews. Two reviewers independently assessed the study quality and extracted relevant data. Statistical heterogeneity among the trials was evaluated by the chi-square and I-square tests. RESULTS: Four randomized controlled trials and two prospective cohort studies involving 18,835 patients met the inclusion criteria. In the meta-analysis, significant differences were observed in knee stability and subjective evaluation with respect to the KT-2000 score (p < .0001), pivot shift test (p = .001), anterior drawer test (p = .0001), Lachman test (p = .0002), subjective International Knee Documentation Committee (IKDC) score (p < .0001), Cincinnati knee score (p = .04), Lysholm score (p = .01), and Tegner score (p = .03). However, the differences in functional assessment in terms of the overall IKDC score (p = .21), range of motion (p = .94), Harner's vertical jump test (p = .09), Daniel's one-leg hop test (p = .50), and complication rate (p = .34) were not significant between the two groups. Failure was reported in two prospective cohort studies in 302 of 14,829 (2%) patients in the autograft group and 157 of 3941 (4%) patients in the irradiated allograft group. CONCLUSION: Irradiated allografts are inferior to autografts for patients undergoing primary ACL reconstruction with respect to knee stability and subjective evaluation. However, no significant differences were found between the two groups in terms of function and complication. The robustness of the findings might need to be further validated because of the limited number of randomized controlled trials. More randomized controlled trials with longer follow-ups are required to further evaluate the failure rate in the two groups.