The effect of preperitoneal pelvic packing for hemodynamically unstable patients with pelvic fractures.

PURPOSE: Hemodynamically unstable patients with pelvic fractures still represent a challenge to trauma surgeons and have a very high mortality. This study was designed to explore the effect of the interventions of direct preperitoneal pelvic packing for the hemodynamically unstable pelvic fractures. METHODS: This retrospective study enrolled 67 cases of severe pelvic fractures with unstable hemodynamics from October 2011 to December 2019. All patients presented in our emergency center and received preperitoneal pelvic packing were included in this study. The indication was persistent systolic blood pressure ≤90 mmHg during initial resuscitation and after transfusion of two units of red blood cells. Patients with hemodynamic stability who need no preperitoneal pelvic packing to control bleeding were excluded. Their demographic characteristics, clinical features, laboratory results, therapeutic interventions, adverse events, and prognostic outcomes were collected from digital information system of electronic medical records. Statistics were described as mean ± standard deviation or medium and analyzed using pair sample t-test or Mann-Whitney U-test. RESULTS: The patients' average age was 41.6 years, ranging from 10 to 88 years. Among them, 45 cases were male (67.2%) and 22 cases were female (32.8%). Significant difference was found regarding the systolic blood pressure (mmHg) in the emergency department (78.4 ± 13.9) and after preperitoneal pelvic packing in the surgery intensive care unit (100.1 ± 17.6) (p < 0.05). Simultaneously, the arterial base deficit (mmol/L) were significantly lower in the surgery intensive care unit (median -6, interquartile range -8 to -2) than in the emergency department (median -10, interquartile range -14 to -8) (p < 0.05). After preperitoneal pelvic packing, 15 patients (22.4%) underwent pelvic angiography for persistent hypotension or suspected ongoing haemorrhage. The overall mortality rate was 29.5% (20 of 67). CONCLUSIONS: Preperitoneal pelvic packing, as a useful surgical technique, is less invasive and can be very efficient in early intra-pelvic bleed control.

Electrostatic confinement and manipulation of DNA molecules for genome analysis.

Very large DNA molecules enable comprehensive analysis of complex genomes, such as human, cancer, and plants because they span across sequence repeats and complex somatic events. When physically manipulated, or analyzed as single molecules, long polyelectrolytes are problematic because of mechanical considerations that include shear-mediated breakage, dealing with the massive size of these coils, or the length of stretched DNAs using common experimental techniques and fluidic devices. Accordingly, we harness analyte "issues" as exploitable advantages by our invention and characterization of the "molecular gate," which controls and synchronizes formation of stretched DNA molecules as DNA dumbbells within nanoslit geometries. Molecular gate geometries comprise micro- and nanoscale features designed to synergize very low ionic strength conditions in ways we show effectively create an "electrostatic bottle." This effect greatly enhances molecular confinement within large slit geometries and supports facile, synchronized electrokinetic loading of nanoslits, even without dumbbell formation. Device geometries were considered at the molecular and continuum scales through computer simulations, which also guided our efforts to optimize design and functionalities. In addition, we show that the molecular gate may govern DNA separations because DNA molecules can be electrokinetically triggered, by varying applied voltage, to enter slits in a size-dependent manner. Lastly, mapping the Mesoplasmaflorum genome, via synchronized dumbbell formation, validates our nascent approach as a viable starting point for advanced development that will build an integrated system capable of large-scale genome analysis.

Single-molecule analysis reveals widespread structural variation in multiple myeloma.

Multiple myeloma (MM), a malignancy of plasma cells, is characterized by widespread genomic heterogeneity and, consequently, differences in disease progression and drug response. Although recent large-scale sequencing studies have greatly improved our understanding of MM genomes, our knowledge about genomic structural variation in MM is attenuated due to the limitations of commonly used sequencing approaches. In this study, we present the application of optical mapping, a single-molecule, whole-genome analysis system, to discover new structural variants in a primary MM genome. Through our analysis, we have identified and characterized widespread structural variation in this tumor genome. Additionally, we describe our efforts toward comprehensive characterization of genome structure and variation by integrating our findings from optical mapping with those from DNA sequencing-based genomic analysis. Finally, by studying this MM genome at two time points during tumor progression, we have demonstrated an increase in mutational burden with tumor progression at all length scales of variation.

Gapless genome assembly of Colletotrichum higginsianum reveals chromosome structure and association of transposable elements with secondary metabolite gene clusters.

BACKGROUND: The ascomycete fungus Colletotrichum higginsianum causes anthracnose disease of brassica crops and the model plant Arabidopsis thaliana. Previous versions of the genome sequence were highly fragmented, causing errors in the prediction of protein-coding genes and preventing the analysis of repetitive sequences and genome architecture. RESULTS: Here, we re-sequenced the genome using single-molecule real-time (SMRT) sequencing technology and, in combination with optical map data, this provided a gapless assembly of all twelve chromosomes except for the ribosomal DNA repeat cluster on chromosome 7. The more accurate gene annotation made possible by this new assembly revealed a large repertoire of secondary metabolism (SM) key genes (89) and putative biosynthetic pathways (77 SM gene clusters). The two mini-chromosomes differed from the ten core chromosomes in being repeat- and AT-rich and gene-poor but were significantly enriched with genes encoding putative secreted effector proteins. Transposable elements (TEs) were found to occupy 7% of the genome by length. Certain TE families showed a statistically significant association with effector genes and SM cluster genes and were transcriptionally active at particular stages of fungal development. All 24 subtelomeres were found to contain one of three highly-conserved repeat elements which, by providing sites for homologous recombination, were probably instrumental in four segmental duplications. CONCLUSION: The gapless genome of C. higginsianum provides access to repeat-rich regions that were previously poorly assembled, notably the mini-chromosomes and subtelomeres, and allowed prediction of the complete SM gene repertoire. It also provides insights into the potential role of TEs in gene and genome evolution and host adaptation in this asexual pathogen.

The Medicago genome provides insight into the evolution of rhizobial symbioses.

Legumes (Fabaceae or Leguminosae) are unique among cultivated plants for their ability to carry out endosymbiotic nitrogen fixation with rhizobial bacteria, a process that takes place in a specialized structure known as the nodule. Legumes belong to one of the two main groups of eurosids, the Fabidae, which includes most species capable of endosymbiotic nitrogen fixation. Legumes comprise several evolutionary lineages derived from a common ancestor 60 million years ago (Myr ago). Papilionoids are the largest clade, dating nearly to the origin of legumes and containing most cultivated species. Medicago truncatula is a long-established model for the study of legume biology. Here we describe the draft sequence of the M. truncatula euchromatin based on a recently completed BAC assembly supplemented with Illumina shotgun sequence, together capturing ∼94% of all M. truncatula genes. A whole-genome duplication (WGD) approximately 58 Myr ago had a major role in shaping the M. truncatula genome and thereby contributed to the evolution of endosymbiotic nitrogen fixation. Subsequent to the WGD, the M. truncatula genome experienced higher levels of rearrangement than two other sequenced legumes, Glycine max and Lotus japonicus. M. truncatula is a close relative of alfalfa (Medicago sativa), a widely cultivated crop with limited genomics tools and complex autotetraploid genetics. As such, the M. truncatula genome sequence provides significant opportunities to expand alfalfa's genomic toolbox.

Tantalum rod implantation for femoral head osteonecrosis: survivorship analysis and determination of prognostic factors for total hip arthroplasty.

PURPOSE: The purpose of this study was to evaluate the moderate survival data of porous tantalum rod implants for the treatment of osteonecrosis of the femoral head (ONFH). Additionally, some independent prognostic factors for conversion to total hip arthroplasty (THA) were identified. METHODS: The porous tantalum rod population was obtained from a prospective, consecutive group of patients treated for Steinberg stage I and II osteonecrosis from April 2009 through July 2011. The historical core decompression and impaction of bone filling particle subjects underwent surgery from April 2007 through March 2009. Surgical data including time of surgery, blood loss, and cell transfusions were recorded. Post-operative values were measured for hospitalization length as well as days requiring a patient-controlled analgesia (PCA) pump. Primary outcomes were Harris hip score and survivorship analysis. Demographics and baseline characteristics included age, sex, etiology, bilateral disease, associated chronic systemic disease, Steinberg stage, Harris hip score, accompanied with bone marrow edema of femoral head, and osteonecrotic lesion size. RESULTS: Demographic/baseline characteristics were similar between two groups. At the post-operative follow-up of 62 months, Harris hip scores were significantly increased (P < 0.0001) when compared to that before surgery in both groups. The magnitude of increase in the tantalum rod implant group was significantly greater than that in the control group (P = 0.0426). With an average follow-up of 48 months (range, 38-62 months), the tantalum rod group had an 84.6 % survival rate. With an average follow-up of 72 months (range, 67-85 months), the control group had a 63.3 % survival rate. A comparison of Kaplan-Meier curves showed significantly higher cumulative survival rates (P = 0.048) for hips with implantation of the porous tantalum rod (74.1 % at 62 months) than for those with impaction composite bone material (49.9 % at 62 months). The Cox proportional-hazard model revealed that implantation of tantalum rod (P = 0.012), bone marrow edema (P = 0.003), corticosteroids intake (P = 0.007), and age less than 50 years (P = 0.014) were the independent prognostic factors related to conversion into THA. CONCLUSIONS: Compared with the traditional impaction composite bone material technique, implantation of tantalum rod in the treatment of Steinberg stages I and II ONFH can obtain better clinical results and higher cumulative survival rates. For patients without the use of corticosteroids, and especially for hips without bone marrow oedema, the clinical results from our study show highly encouraging survival rates and a delay in or prevention of conversion into THA.

A clone-free, single molecule map of the domestic cow (Bos taurus) genome.

BACKGROUND: The cattle (Bos taurus) genome was originally selected for sequencing due to its economic importance and unique biology as a model organism for understanding other ruminants, or mammals. Currently, there are two cattle genome sequence assemblies (UMD3.1 and Btau4.6) from groups using dissimilar assembly algorithms, which were complemented by genetic and physical map resources. However, past comparisons between these assemblies revealed substantial differences. Consequently, such discordances have engendered ambiguities when using reference sequence data, impacting genomic studies in cattle and motivating construction of a new optical map resource--BtOM1.0--to guide comparisons and improvements to the current sequence builds. Accordingly, our comprehensive comparisons of BtOM1.0 against the UMD3.1 and Btau4.6 sequence builds tabulate large-to-immediate scale discordances requiring mediation. RESULTS: The optical map, BtOM1.0, spanning the B. taurus genome (Hereford breed, L1 Dominette 01449) was assembled from an optical map dataset consisting of 2,973,315 (439 X; raw dataset size before assembly) single molecule optical maps (Rmaps; 1 Rmap = 1 restriction mapped DNA molecule) generated by the Optical Mapping System. The BamHI map spans 2,575.30 Mb and comprises 78 optical contigs assembled by a combination of iterative (using the reference sequence: UMD3.1) and de novo assembly techniques. BtOM1.0 is a high-resolution physical map featuring an average restriction fragment size of 8.91 Kb. Comparisons of BtOM1.0 vs. UMD3.1, or Btau4.6, revealed that Btau4.6 presented far more discordances (7,463) vs. UMD3.1 (4,754). Overall, we found that Btau4.6 presented almost double the number of discordances than UMD3.1 across most of the 6 categories of sequence vs. map discrepancies, which are: COMPLEX (misassembly), DELs (extraneous sequences), INSs (missing sequences), ITs (Inverted/Translocated sequences), ECs (extra restriction cuts) and MCs (missing restriction cuts). CONCLUSION: Alignments of UMD3.1 and Btau4.6 to BtOM1.0 reveal discordances commensurate with previous reports, and affirm the NCBI's current designation of UMD3.1 sequence assembly as the "reference assembly" and the Btau4.6 as the "alternate assembly." The cattle genome optical map, BtOM1.0, when used as a comprehensive and largely independent guide, will greatly assist improvements to existing sequence builds, and later serve as an accurate physical scaffold for studies concerning the comparative genomics of cattle breeds.

Prediction of survival prognosis after surgery in patients with symptomatic metastatic spinal cord compression from non-small cell lung cancer.

BACKGROUND: The aim of this study was to develop a scoring system for prediction of survival prognosis after surgery in patients with symptomatic metastatic spinal cord compression (MSCC) from non-small cell lung cancer (NSCLC). METHODS: We retrospectively analyzed nine preoperative characteristics for survival in a series of 64 patients with NSCLC who were operated with posterior decompression and spine stabilization for MSCC. Characteristics significantly associated with survival on multivariate analysis were included in the scoring system. The scoring point for each significant characteristic was derived from the hazard ratios on Cox proportional hazards model. The total score for each patient was obtained by adding the scoring points of all significant characteristics. RESULTS: Eastern Cooperative Oncology Group (ECOG) performance status, number of involved vertebrae, visceral metastases, and time developing motor deficits had significant impact on survival on multivariate analysis and were included in the scoring system. According to the prognostic scores, which ranged from 4 to 10 points, three prognostic groups were designed: 4-5 points (n = 22), 6-7 points (n = 23), and 8-10 points (n = 19). The corresponding 6-month survival rates were 95, 47 and 11%, respectively (P < 0.0001). In addition, the functional outcome was worse in the group of patients with 8-10 points compared with other two prognostic groups. CONCLUSIONS: The new scoring system will enable physicians to identify patient with MSCC from NSCLC who may be a candidate for decompression and spine stabilization, more radical surgery, or supportive care alone. Patients with scores of 4-5, who have the most favorable survival prognosis and functional outcome, can be treated with more radical surgery in order to realize better local control of disease and prevent the occurrence of local disease. Patients with scores of 6-7 points should be surgical candidates, because survival prognosis and functional outcome are acceptable after surgery, while patients with scores of 8-10 points, who have the shortest survival time and poorest functional outcome after surgery, appear to be best treated with radiotherapy or best supportive care.

An improved genome release (version Mt4.0) for the model legume Medicago truncatula.

BACKGROUND: Medicago truncatula, a close relative of alfalfa, is a preeminent model for studying nitrogen fixation, symbiosis, and legume genomics. The Medicago sequencing project began in 2003 with the goal to decipher sequences originated from the euchromatic portion of the genome. The initial sequencing approach was based on a BAC tiling path, culminating in a BAC-based assembly (Mt3.5) as well as an in-depth analysis of the genome published in 2011. RESULTS: Here we describe a further improved and refined version of the M. truncatula genome (Mt4.0) based on de novo whole genome shotgun assembly of a majority of Illumina and 454 reads using ALLPATHS-LG. The ALLPATHS-LG scaffolds were anchored onto the pseudomolecules on the basis of alignments to both the optical map and the genotyping-by-sequencing (GBS) map. The Mt4.0 pseudomolecules encompass ~360 Mb of actual sequences spanning 390 Mb of which ~330 Mb align perfectly with the optical map, presenting a drastic improvement over the BAC-based Mt3.5 which only contained 70% sequences (~250 Mb) of the current version. Most of the sequences and genes that previously resided on the unanchored portion of Mt3.5 have now been incorporated into the Mt4.0 pseudomolecules, with the exception of ~28 Mb of unplaced sequences. With regard to gene annotation, the genome has been re-annotated through our gene prediction pipeline, which integrates EST, RNA-seq, protein and gene prediction evidences. A total of 50,894 genes (31,661 high confidence and 19,233 low confidence) are included in Mt4.0 which overlapped with ~82% of the gene loci annotated in Mt3.5. Of the remaining genes, 14% of the Mt3.5 genes have been deprecated to an "unsupported" status and 4% are absent from the Mt4.0 predictions. CONCLUSIONS: Mt4.0 and its associated resources, such as genome browsers, BLAST-able datasets and gene information pages, can be found on the JCVI Medicago web site (http://www.jcvi.org/medicago). The assembly and annotation has been deposited in GenBank (BioProject: PRJNA10791). The heavily curated chromosomal sequences and associated gene models of Medicago will serve as a better reference for legume biology and comparative genomics.

[A modified porous tantalum implant technology for osteonecrosis of the femoral head: survival and prognostic evaluations of conversion into total hip arthroplasty].

OBJECTIVE: To assess the survival and prognostic significance of various demographic and radiographic parameters for conversion into total hip arthroplasty after treatment with a modified porous tantalum implant technology for early and intermediate stages of osteonecrosis of the femoral head (ONFH). METHODS: This study included 45 patients (59 hips) with Steinberg Stage I-IV A ONFH undergoing progressively core decompression, impaction bone grafting of 5 mm-composite bone filling material and inserting of a porous tantalum implant. Weight-bearing was forbidden within the first 3 months after implants. RESULTS: A total of 57 hips (44 patients) were available during a mean follow-up period of 44.8 (11-62) months. Their mean age was 43 (21-70) years. The mean Harris hip score significantly improved from 59.93 ± 2.80 preoperative to 77.84 ± 2.95 at the last follow-up (P < 0.001). Overall, 11 hips (19.30%) were converted into total hip arthroplasty. The overall survival rate was 72.49% at 60 months postoperatively. The Cox proportional hazard model revealed that bone marrow edema was an independent prognostic factor related with a conversion into total hip arthroplasty. CONCLUSION: Higher survival rates may be obtained from modified tantalum implant technology for early and intermediate stages of ONFH. And prognosis was poor for patients of ONFH with bone marrow edema.

The effect of carbon nanoparticles vs. immune colloidal gold technique test strips on parathyroid protection in total thyroidectomy: A randomized clinical trial study.

BACKGROUND: The long-term effect of intraoperative usage of carbon nanoparticles (CN) and parathyroid hormone (PTH) test strip using immune colloidal gold technique (ICGT) is unclear. This study aims to compare the effect of intraoperative usage of CN and ICGT test strips on PG function. METHODS: This randomized clinical study involved adult patients who underwent total thyroidectomy. They were randomly allocated into three groups (control, CN, and ICGT group). Clinical data were analyzed. RESULTS: Each group involved 98 patients. Serum calcium and PTH concentrations at 24 h postoperatively (PTH24h) were higher in CN group. The parathyroid function recovered quicker in CN group. Use of CN increased in situ PG preservation and PTH24h. Mediation analysis indicated that 23.05% of the total effect of CN on PTH24h was attributed to PGRIS. CONCLUSION: CN holds promise to improve in situ PG preservation and protect PG vasculature, thereby reducing the incidence of early hypoparathyroidism. The value of ICGT test strips for PG protection is dubious.

Targeted phasing of 2-200 kilobase DNA fragments with a short-read sequencer and a single-tube linked-read library method.

In the human genome, heterozygous sites refer to genomic positions with a different allele or nucleotide variant on the maternal and paternal chromosomes. Resolving these allelic differences by chromosomal copy, also known as phasing, is achievable on a short-read sequencer when using a library preparation method that captures long-range genomic information. TELL-Seq is a library preparation that captures long-range genomic information with the aid of molecular identifiers (barcodes). The same barcode is used to tag the reads derived from the same long DNA fragment within a range of up to 200 kilobases (kb), generating linked-reads. This strategy can be used to phase an entire genome. Here, we introduce a TELL-Seq protocol developed for targeted applications, enabling the phasing of enriched loci of varying sizes, purity levels, and heterozygosity. To validate this protocol, we phased 2-200 kb loci enriched with different methods: CRISPR/Cas9-mediated excision coupled with pulse-field electrophoresis for the longest fragments, CRISPR/Cas9-mediated protection from exonuclease digestion for mid-size fragments, and long PCR for the shortest fragments. All selected loci have known clinical relevance: BRCA1, BRCA2, MLH1, MSH2, MSH6, APC, PMS2, SCN5A-SCN10A, and PKI3CA. Collectively, the analyses show that TELL-Seq can accurately phase 2-200 kb targets using a short-read sequencer.

Discovery of structural alterations in solid tumor oligodendroglioma by single molecule analysis.

BACKGROUND: Solid tumors present a panoply of genomic alterations, from single base changes to the gain or loss of entire chromosomes. Although aberrations at the two extremes of this spectrum are readily defined, comprehensive discernment of the complex and disperse mutational spectrum of cancer genomes remains a significant challenge for current genome analysis platforms. In this context, high throughput, single molecule platforms like Optical Mapping offer a unique perspective. RESULTS: Using measurements from large ensembles of individual DNA molecules, we have discovered genomic structural alterations in the solid tumor oligodendroglioma. Over a thousand structural variants were identified in each tumor sample, without any prior hypotheses, and often in genomic regions deemed intractable by other technologies. These findings were then validated by comprehensive comparisons to variants reported in external and internal databases, and by selected experimental corroborations. Alterations range in size from under 5 kb to hundreds of kilobases, and comprise insertions, deletions, inversions and compound events. Candidate mutations were scored at sub-genic resolution and unambiguously reveal structural details at aberrant loci. CONCLUSIONS: The Optical Mapping system provides a rich description of the complex genomes of solid tumors, including sequence level aberrations, structural alterations and copy number variants that power generation of functional hypotheses for oligodendroglioma genetics.

Improving mammalian genome scaffolding using large insert mate-pair next-generation sequencing.

BACKGROUND: Paired-tag sequencing approaches are commonly used for the analysis of genome structure. However, mammalian genomes have a complex organization with a variety of repetitive elements that complicate comprehensive genome-wide analyses. RESULTS: Here, we systematically assessed the utility of paired-end and mate-pair (MP) next-generation sequencing libraries with insert sizes ranging from 170 bp to 25 kb, for genome coverage and for improving scaffolding of a mammalian genome (Rattus norvegicus). Despite a lower library complexity, large insert MP libraries (20 or 25 kb) provided very high physical genome coverage and were found to efficiently span repeat elements in the genome. Medium-sized (5, 8 or 15 kb) MP libraries were much more efficient for genome structure analysis than the more commonly used shorter insert paired-end and 3 kb MP libraries. Furthermore, the combination of medium- and large insert libraries resulted in a 3-fold increase in N50 in scaffolding processes. Finally, we show that our data can be used to evaluate and improve contig order and orientation in the current rat reference genome assembly. CONCLUSIONS: We conclude that applying combinations of mate-pair libraries with insert sizes that match the distributions of repetitive elements improves contig scaffolding and can contribute to the finishing of draft genomes.

High-resolution human genome structure by single-molecule analysis.

Variation in genome structure is an important source of human genetic polymorphism: It affects a large proportion of the genome and has a variety of phenotypic consequences relevant to health and disease. In spite of this, human genome structure variation is incompletely characterized due to a lack of approaches for discovering a broad range of structural variants in a global, comprehensive fashion. We addressed this gap with Optical Mapping, a high-throughput, high-resolution single-molecule system for studying genome structure. We used Optical Mapping to create genome-wide restriction maps of a complete hydatidiform mole and three lymphoblast-derived cell lines, and we validated the approach by demonstrating a strong concordance with existing methods. We also describe thousands of new variants with sizes ranging from kb to Mb.

[Efficacies of bone-marrow-derived mononuclear cells with a hydroxylapatite composite in the treatment of osteonecrosis of the femoral head].

OBJECTIVE: To evaluate the efficacies of core decompression and implantation of concentrated autologous bone marrow containing mononuclear cells (BMMCs) with porous hydroxylapatite composite in the treatment of osteonecrosis of the femoral head. METHODS: A total of 35 patients with 57 osteonecrosis hips with ARCO stage I, stage II and stage IIIA disease were treated by BMMCs with a porous hydroxylapatite composite. The mean age at surgery was 39.4 (26-58) years and the mean period of follow-up 28 (12-40) months. In the control group, cell-free porous hydroxylapatite composite was implanted into 17 patients (27 hips) with osteonecrosis of the femoral head and the outcomes were compared. RESULTS: At the last follow-up, postoperative Harris hip scores significantly increased in both groups (P < 0.0001). The magnitude of increase was significantly greater in the BMMCs group compared with the control group (28.3% ± 0.9% vs 18.4% ± 1.7%, P < 0.01). Postoperative visual analog scale (VAS) scores significantly decreased in both groups (P < 0.01). The magnitude of decrease was significantly greater in the BMMCs group compared with the control group (-70.2% ± 2.1% vs -51.7% ± 2.9%, P < 0.001). The clinical success rate was significantly higher in the BMMCs group compared with the control group (75.4% vs 37.0%, P < 0.01). The radiological success rates were similar between the BMMCs and control groups (59.6% vs 40.7%, P = 0.1046). CONCLUSION: The combined regimen of core decompression and implantation of concentrated autologous BMMCs with porous hydroxylapatite composite appears to confer benefits in the treatment of in stages I-IIIA osteonecrosis of the femoral head.

Early effect of standalone oblique lateral interbody fusion vs. combined with lateral screw fixation of the vertebral body on single-level lumbar degenerative disc disease: A pilot study.

BACKGROUND: For the treatment of single-level lumbar degenerative disc disease (DDD), oblique lateral interbody fusion (OLIF) has clinical advantages. Whether internal fixation needs to be combined for treatment has been the subject of debate. OBJECTIVE: To compare the early clinical effects of standalone oblique lateral interbody fusion (S-OLIF) versus OLIF combined with lateral screw fixation of the vertebral body (F-OLIF) on single-level lumbar DDD. METHODS: A retrospective analysis was performed on the data of 34 patients for whom the OLIF technique was applied to treat single-level lumbar DDD from August 2018 to May 2021. Patients were divided into the S-OLIF (n= 18) and F-OLIF groups (n= 16). Intraoperative blood loss, operative time, and length of hospital stay were recorded. The pain visual analogue scale (VAS) and Oswestry disability index (ODI) before and after the operation were evaluated. The disc height (DH), foraminal height (FH), fused segment lordosis (FSL), lumbar lordosis (LL), cage subsidence, and fusion by CT examination were measured before and after the operation. RESULTS: The S-OLIF group experienced a shorter operative time and less intraoperative blood loss than the F-OLIF group, and the differences were statistically significant (p< 0.05), but the difference in the length of hospital stay was not statistically significant. The postoperative VAS score and ODI of the two groups were significantly lower than those before the operation, but the postoperative differences between the two groups were not statistically significant. Differences were not statistically significant in postoperative FH, DH, FSL and LL of the two groups. Both groups were followed up for no less than 12 months. In the two groups, fusion was achieved at the last follow-up visit. CONCLUSION: According to short-term follow-up results, both S-OLIF and F-OLIF can achieve reliable and stable fusion and good clinical effect in the treatment of single-level lumbar DDD.

Targeted Phasing of 2-200 Kilobase DNA Fragments with a Short-Read Sequencer and a Single-Tube Linked-Read Library Method.

In the human genome, heterozygous sites are genomic positions with different alleles inherited from each parent. On average, there is a heterozygous site every 1-2 kilobases (kb). Resolving whether two alleles in neighboring heterozygous positions are physically linked-that is, phased-is possible with a short-read sequencer if the sequencing library captures long-range information. TELL-Seq is a library preparation method based on millions of barcoded micro-sized beads that enables instrument-free phasing of a whole human genome in a single PCR tube. TELL-Seq incorporates a unique molecular identifier (barcode) to the short reads generated from the same high-molecular-weight (HMW) DNA fragment (known as 'linked-reads'). However, genome-scale TELL-Seq is not cost-effective for applications focusing on a single locus or a few loci. Here, we present an optimized TELL-Seq protocol that enables the cost-effective phasing of enriched loci (targets) of varying sizes, purity levels, and heterozygosity. Targeted TELL-Seq maximizes linked-read efficiency and library yield while minimizing input requirements, fragment collisions on microbeads, and sequencing burden. To validate the targeted protocol, we phased seven 180-200 kb loci enriched by CRISPR/Cas9-mediated excision coupled with pulse-field electrophoresis, four 20 kb loci enriched by CRISPR/Cas9-mediated protection from exonuclease digestion, and six 2-13 kb loci amplified by PCR. The selected targets have clinical and research relevance (BRCA1, BRCA2, MLH1, MSH2, MSH6, APC, PMS2, SCN5A-SCN10A, and PKI3CA). These analyses reveal that targeted TELL-Seq provides a reliable way of phasing allelic variants within targets (2-200 kb in length) with the low cost and high accuracy of short-read sequencing.

AGORA: Assembly Guided by Optical Restriction Alignment.

BACKGROUND: Genome assembly is difficult due to repeated sequences within the genome, which create ambiguities and cause the final assembly to be broken up into many separate sequences (contigs). Long range linking information, such as mate-pairs or mapping data, is necessary to help assembly software resolve repeats, thereby leading to a more complete reconstruction of genomes. Prior work has used optical maps for validating assemblies and scaffolding contigs, after an initial assembly has been produced. However, optical maps have not previously been used within the genome assembly process. Here, we use optical map information within the popular de Bruijn graph assembly paradigm to eliminate paths in the de Bruijn graph which are not consistent with the optical map and help determine the correct reconstruction of the genome. RESULTS: We developed a new algorithm called AGORA: Assembly Guided by Optical Restriction Alignment. AGORA is the first algorithm to use optical map information directly within the de Bruijn graph framework to help produce an accurate assembly of a genome that is consistent with the optical map information provided. Our simulations on bacterial genomes show that AGORA is effective at producing assemblies closely matching the reference sequences.Additionally, we show that noise in the optical map can have a strong impact on the final assembly quality for some complex genomes, and we also measure how various characteristics of the starting de Bruijn graph may impact the quality of the final assembly. Lastly, we show that a proper choice of restriction enzyme for the optical map may substantially improve the quality of the final assembly. CONCLUSIONS: Our work shows that optical maps can be used effectively to assemble genomes within the de Bruijn graph assembly framework. Our experiments also provide insights into the characteristics of the mapping data that most affect the performance of our algorithm, indicating the potential benefit of more accurate optical mapping technologies, such as nano-coding.

Genome sequencing of ovine isolates of Mycobacterium avium subspecies paratuberculosis offers insights into host association.

BACKGROUND: The genome of Mycobacterium avium subspecies paratuberculosis (MAP) is remarkably homogeneous among the genomes of bovine, human and wildlife isolates. However, previous work in our laboratories with the bovine K-10 strain has revealed substantial differences compared to sheep isolates. To systematically characterize all genomic differences that may be associated with the specific hosts, we sequenced the genomes of three U.S. sheep isolates and also obtained an optical map. RESULTS: Our analysis of one of the isolates, MAP S397, revealed a genome 4.8 Mb in size with 4,700 open reading frames (ORFs). Comparative analysis of the MAP S397 isolate showed it acquired approximately 10 large sequence regions that are shared with the human M. avium subsp. hominissuis strain 104 and lost 2 large regions that are present in the bovine strain. In addition, optical mapping defined the presence of 7 large inversions between the bovine and ovine genomes (~ 2.36 Mb). Whole-genome sequencing of 2 additional sheep strains of MAP (JTC1074 and JTC7565) further confirmed genomic homogeneity of the sheep isolates despite the presence of polymorphisms on the nucleotide level. CONCLUSIONS: Comparative sequence analysis employed here provided a better understanding of the host association, evolution of members of the M. avium complex and could help in deciphering the phenotypic differences observed among sheep and cattle strains of MAP. A similar approach based on whole-genome sequencing combined with optical mapping could be employed to examine closely related pathogens. We propose an evolutionary scenario for M. avium complex strains based on these genome sequences.