Identification and Biotechnical Potential of a Gcn5-Related N-Acetyltransferase Gene in Enhancing Microalgal Biomass and Starch Production.

Microalgae are promising feedstocks for starch production, which are precursors for bioenergy and chemicals manufacturing. Though starch biosynthesis has been intensively studied in the green alga Chlamydomonas reinhardtii, regulatory mechanisms governing starch metabolism in this model species have remained largely unknown to date. We proposed that altering triacylglycerol (TAG) biosynthesis may trigger intrinsic regulatory pathways governing starch metabolism. In accordance with the hypothesis, it was observed in this study that overexpression of the plastidial lysophosphatidic acid acyltransferase gene (i.e. LPAAT1) in C. reinhardtii significantly enhanced TAG biosynthesis under nitrogen (N)-replete conditions, whereas the starch biosynthesis was enhanced in turn under N depletion. By the exploitation of transcriptomics analysis, a putative regulatory gene coding Gcn5-related N-acetyltransferase (GNAT19) was identified, which was up-regulated by 11-12 times in the CrLPAAT1 OE lines. Overexpression of the cloned full-length CrGNAT19 cDNA led to significant increase in the starch content of C. reinhardtii cells grown under both N-replete and N-depleted conditions, which was up to 4 times and 26.7% higher than that of the empty vector control, respectively. Moreover, the biomass yield of the CrGNAT19 OE lines reached 1.5 g L-1 after 2 days under N-depleted conditions, 72% higher than that of the empty vector control (0.87 g L-1). Overall, the yield of starch increased by 118.5% in CrGNAT19 OE lines compared to that of the control. This study revealed the great biotechnical potentials of an unprecedented GNAT19 gene in enhancing microalgal starch and biomass production.

Postoperative delirium after hip surgery is a potential risk factor for incident dementia: A systematic review and meta-analysis of prospective studies.

BACKGROUND: Although a few trials have explored the relationship between postoperative delirium (POD) and incident dementia in patients with hip surgery, the numbers of participants in each study are relatively small. Thus, we performed a meta-analysis to examine whether POD after hip surgery is a risk factor for incident dementia. METHODS: Six prospective cohort studies investigating the development of incident dementia in patients with POD after hip surgery were retrieved from PubMed, Embase, and the Cochrane Library. We performed a pairwise meta-analysis using fixed- and random- effect models. RESULTS: POD significantly increased the risk of incident dementia and cognitive decline (overall odds ratio [ORs] = 8.957; 95 % confidence interval [CI], 5.444-14.737; P <  0.001 in fixed-effects model; overall ORs = 8.962; 95 % CI, 5.344-15.029; P <  0.001 in random-effects model). A publication bias was not evident in this study. CONCLUSIONS: Our meta-analysis revealed that POD after hip surgery is a risk factor for incident dementia. Early identification of cognitive function should be needed after surgery and appropriate prevention and treatment for dementia will be required, especially in cases with POD.

Biosynthesis of Triacylglycerol Molecules with a Tailored PUFA Profile in Industrial Microalgae.

The composition of polyunsaturated fatty acids (PUFAs) in triacylglycerols (TAGs) is key to health benefits and for oil applications, yet the underlying genetic mechanism remains poorly understood. In this study, by in silico, ex vivo, and in vivo profiling of type-2 diacylglycerol acyltransferases (DGAT2s) in Nannochloropsis oceanica we revealed two novel PUFA-preferring enzymes that discriminate individual PUFA species in TAG assembly, with NoDGAT2J for linoleic acid (LA) and NoDGAT2K for eicosapentaenoic acid (EPA). The LA and EPA composition of TAG molecules is mediated in vivo via the functional partitioning between NoDGAT2J and 2K, both of which are localized in the chloroplast envelope. By modulating transcript abundance of the DGAT2s, an N. oceanica strain bank was created, where proportions of LA and EPA in TAG vary by 18.7-fold (between 0.21% and 3.92% dry weight) and 34.7-fold (between 0.09% and 3.12% dry weight), respectively. These findings lay the foundation for producing designer TAG molecules with tailored health benefits or for biofuel applications in industrial microalgae and higher-plant crops.

Arthroscopic treatment of psoas abscess concurrent with septic arthritis of the hip joint.

PURPOSE: To describe the outcomes of 7 cases of psoas abscess concurrent with septic arthritis of the hip treated by hip arthroscopy alone. METHODS: We retrospectively collected the data of patients who underwent arthroscopic drainage of psoas abscess concurrent with septic arthritis of the hip. Arthroscopic debridement was performed in both the central and peripheral hip joint compartments. In all cases, the iliopsoas compartment was accessed from the peripheral compartment through an anterior capsulotomy without limb traction. After debridement and drainage of the iliopsoas compartment, a suction drain tube was placed in the iliopsoas compartment through an enlarged anterior capsulotomy and another tube in the peripheral compartment. Postoperative intravenous antibiotics were administered on the basis of culture results; in cases with no positive culture, empirical antibiotics were administered for 4 to 6 weeks after surgery. RESULTS: 7 patients underwent arthroscopic debridement and drainage for a psoas abscess concurrent with hip joint septic arthritis. Laboratory tests were normalized within 4 weeks after hip arthroscopy in all patients. At a median follow-up of 16 months (range, 13-30 months) after surgery, infection recurrence was absent in all patients. CONCLUSIONS: Arthroscopic debridement alone could be an effective treatment alternative to open surgery for psoas abscess concurrent with hip joint septic arthritis.

Characterization of type 2 diacylglycerol acyltransferases in Chlamydomonas reinhardtii reveals their distinct substrate specificities and functions in triacylglycerol biosynthesis.

Diacylglycerol acyltransferases (DGATs) catalyze a rate-limiting step of triacylglycerol (TAG) biosynthesis in higher plants and yeast. The genome of the green alga Chlamydomonas reinhardtii has multiple genes encoding type 2 DGATs (DGTTs). Here we present detailed functional and biochemical analyses of Chlamydomonas DGTTs. In vitro enzyme analysis using a radiolabel-free assay revealed distinct substrate specificities of three DGTTs: CrDGTT1 preferred polyunsaturated acyl CoAs, CrDGTT2 preferred monounsaturated acyl CoAs, and CrDGTT3 preferred C16 CoAs. When diacylglycerol was used as the substrate, CrDGTT1 preferred C16 over C18 in the sn-2 position of the glycerol backbone, but CrDGTT2 and CrDGTT3 preferred C18 over C16. In vivo knockdown of CrDGTT1, CrDGTT2 or CrDGTT3 resulted in 20-35% decreases in TAG content and a reduction of specific TAG fatty acids, in agreement with the findings of the in vitro assay and fatty acid feeding test. These results demonstrate that CrDGTT1, CrDGTT2 and CrDGTT3 possess distinct specificities toward acyl CoAs and diacylglycerols, and may work in concert spatially and temporally to synthesize diverse TAG species in C. reinhardtii. CrDGTT1 was shown to prefer prokaryotic lipid substrates and probably resides in both the endoplasmic reticulum and chloroplast envelope, indicating its role in prokaryotic and eukaryotic TAG biosynthesis. Based on these findings, we propose a working model for the role of CrDGTT1 in TAG biosynthesis. This work provides insight into TAG biosynthesis in C. reinhardtii, and paves the way for engineering microalgae for production of biofuels and high-value bioproducts.

Nannochloropsis genomes reveal evolution of microalgal oleaginous traits.

Oleaginous microalgae are promising feedstock for biofuels, yet the genetic diversity, origin and evolution of oleaginous traits remain largely unknown. Here we present a detailed phylogenomic analysis of five oleaginous Nannochloropsis species (a total of six strains) and one time-series transcriptome dataset for triacylglycerol (TAG) synthesis on one representative strain. Despite small genome sizes, high coding potential and relative paucity of mobile elements, the genomes feature small cores of ca. 2,700 protein-coding genes and a large pan-genome of >38,000 genes. The six genomes share key oleaginous traits, such as the enrichment of selected lipid biosynthesis genes and certain glycoside hydrolase genes that potentially shift carbon flux from chrysolaminaran to TAG synthesis. The eleven type II diacylglycerol acyltransferase genes (DGAT-2) in every strain, each expressed during TAG synthesis, likely originated from three ancient genomes, including the secondary endosymbiosis host and the engulfed green and red algae. Horizontal gene transfers were inferred in most lipid synthesis nodes with expanded gene doses and many glycoside hydrolase genes. Thus multiple genome pooling and horizontal genetic exchange, together with selective inheritance of lipid synthesis genes and species-specific gene loss, have led to the enormous genetic apparatus for oleaginousness and the wide genomic divergence among present-day Nannochloropsis. These findings have important implications in the screening and genetic engineering of microalgae for biofuels.

Revision of ceramic head fracture after third generation ceramic-on-ceramic total hip arthroplasty.

We performed 24 revisions of fractures of third generation ceramic heads. The stem was not changed in 20 revisions; a new ceramic-on-ceramic bearing was used in four and a metal-on-polyethylene bearing in 16. The stem was changed in four revisions; a new ceramic-on-ceramic bearing was used in three and a metal-on-polyethylene bearing in one. During the follow-up of 57.5 months, complications occurred in five hips among the 20 stem retained revisions: a fracture of the new ceramic head in two, metallosis with pseudocyst in two, and femoral osteolysis with stem loosening in one. However, there were no complications in the four revisions where the stem was changed. Revision surgery after ceramic head fracture shows high rates of early complications. We recommend stem revision in cases of THA failure due to fracture of a modern ceramic head.

Phospholipid:diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii.

Many unicellular microalgae produce large amounts (∼20 to 50% of cell dry weight) of triacylglycerols (TAGs) under stress (e.g., nutrient starvation and high light), but the synthesis and physiological role of TAG are poorly understood. We present detailed genetic, biochemical, functional, and physiological analyses of phospholipid:diacylglycerol acyltransferase (PDAT) in the green microalga Chlamydomonas reinhardtii, which catalyzes TAG synthesis via two pathways: transacylation of diacylglycerol (DAG) with acyl groups from phospholipids and galactolipids and DAG:DAG transacylation. We demonstrate that PDAT also possesses acyl hydrolase activities using TAG, phospholipids, galactolipids, and cholesteryl esters as substrates. Artificial microRNA silencing of PDAT in C. reinhardtii alters the membrane lipid composition, reducing the maximum specific growth rate. The data suggest that PDAT-mediated membrane lipid turnover and TAG synthesis is essential for vigorous growth under favorable culture conditions and for membrane lipid degradation with concomitant production of TAG for survival under stress. The strong lipase activity of PDAT with broad substrate specificity suggests that this enzyme could be a potential biocatalyst for industrial lipid hydrolysis and conversion, particularly for biofuel production.

Isolation and characterization of a sex-specific lectin in a marine red alga, Aglaothamnion oosumiense Itono.

In red algae, spermatial binding to female trichogynes is mediated by a lectin-carbohydrate complementary system. Aglaothamnion oosumiense is a microscopic filamentous red alga. The gamete recognition and binding occur at the surface of the hairlike trichogyne on the female carpogonium. Male spermatia are nonmotile. Previous studies suggested the presence of a lectin responsible for gamete recognition on the surface of female trychogynes. A novel N-acetyl-D-galactosamine-specific protein was isolated from female plants of A. oosumiense by affinity chromatography and named AOL1. The lectin was monomeric and did not agglutinate horse blood or human erythrocytes. The N-terminal amino acid sequence of the protein was analyzed, and degenerate primers were designed. A full-length cDNA encoding the lectin was obtained using rapid amplification of cDNA ends-PCR (RACE-PCR). The cDNA was 1,095 bp in length and coded for a protein of 259 amino acids with a deduced molecular mass of 21.4 kDa, which agreed well with the protein data. PCR analysis using genomic DNA showed that both male and female plants have this gene. However, Northern blotting and two-dimensional electrophoresis showed that this protein was expressed 12 to 15 times more in female plants. The lectin inhibited spermatial binding to the trichogynes when preincubated with spermatia, suggesting its involvement in gamete binding.

Differences in donor CXCR4 expression levels are correlated with functional capacity and therapeutic outcome of angiogenic treatment with endothelial colony forming cells.

CXCR4 expression is important for cell migration and recruitment, suggesting that the expression levels of CXCR4 may be correlated with functional activity of implanted cells for therapeutic neovascularization. Here, we examined differences between umbilical cord blood (CB) donors in the CXCR4 levels of endothelial colony forming cells (ECFCs), which are a subtype of endothelial progenitor cells (EPCs). We investigated the relationships between CXCR4 expression level and SDF-1alpha-induced vascular properties in vitro, and their in vivo contributions to neovascularization. We found that ECFCs isolated from different donors showed differences in CXCR4 expression that were linearly correlated with SDF-1alpha-induced migratory capacity. ECFCs with high CXCR4 expression showed enhanced ERK and Akt activation in response to SDF-1alpha. In addition, SDF-1alpha-induced migration and ERK1/2, Akt, and eNOS activation were reduced by AMD3100, a CXCR4-specific peptide antagonist, or by siRNA-CXCR4. Administration of high-CXCR4-expressing ECFCs resulted in a significant increase in therapeutic potential for blood flow recovery, tissue healing and capillary density compared to low-CXCR4-expressing ECFCs in hindlimb ischemia. Taken together, the functional differences among ECFCs derived from different donors depended on the level of CXCR4 expression, suggesting that CXCR4 expression levels in ECFCs could be a predictive marker for success of ECFC-based angiogenic therapy.

Mesenchymal stem-cell transplantation for hypoxic-ischemic brain injury in neonatal rat model.

Neonatal hypoxic-ischemic brain injury (HIE) remains a major cause of neurologic disabilities. However, many experimental therapies have shown limited successes. We assessed whether human mesenchymal stem cells (MSCs) could be transplanted in the HIE rat brain to improve neurologic disabilities. P7 SD rats were either subjected to left carotid artery ligation and hypoxic exposure [hypoxia-ischemia (HI)] or sham operation and normoxic exposure (sham). On P10, rat pubs received either PKH26-labeled MSCs or buffer via intracardial injection, resulting in four experimental groups: sham-buffer, sham-MSC, HI-buffer, and HI-MSC. Cylinder test and accelerating rotarod test were performed 14, 20, 30, and 40 d after injection. Six weeks after injection, cresyl violet and double immunofluorescence staining were performed. MSCs were transplanted to the whole brain mainly after HI. Glial fibrillary acidic protein and OX42 were more abundantly colocalized with MSC than neuronal specific nuclear protein or myelin basic protein. There were no significant differences in the total amounts and cell types between the lesioned and nonlesioned hemisphere. The lesioned hemispheric volume was decreased after HI (p = 0.012) but not restored by MSC. Neurologic performance was significantly impaired only on the cylinder test after HI (p = 0.034), and MSC transplants improved it (p = 0.010). These suggest MSC can be a candidate for the treatment of neonatal HIE.

Fetal mesenchymal stem cells derived from human umbilical cord sustain primitive characteristics during extensive expansion.

Stem cells of fetal origin lie between embryonic and adult stem cells in terms of potentiality. Because of the ethical controversy surrounding embryonic stem cells and the relatively inferior quality of adult stem cells, the use of fetal stem cells would be an attractive option in future therapeutic applications. Here, we have investigated primitive characteristics of human umbilical-cord-derived fetal mesenchymal stem cells (UC fMSCs) during extensive expansion. We have successfully isolated and cultured UC fMSCs from all UC samples, but with two early fungal contaminations. UC fMSCs proliferated without significant evidence of morphological changes, and the average cumulative population-doubling level was over 25 for about 3 months. UC fMSCs showed the positive expression of several CD markers, known to be related to MSCs, including CD73 (SH-3, 4), CD90 (Thy-1), CD105 (SH-2), CD117 (c-kit), and CD166 (ALCAM). They demonstrated primitive properties throughout the expansion period: multilineage differentiation potentials examined by functional assays, a variety of pluripotent stem cell markers including Nanog, Oct-4, Sox-2, Rex-1, SSEA-3, SSEA-4, Tra-1-60, and Tra-1-81, minimal evidence of senescence as shown by beta-galactosidase staining, and the consistent expression of telomerase activity. These results suggest that UC fMSCs have more primitive properties than adult MSCs, which might make them a useful source of MSCs for clinical applications.

Comparative study of unilateral and bilateral cages with respect to clinical outcomes and stability in instrumented posterior lumbar interbody fusion.

OBJECTIVE: We sought to compare the clinical and radiological results of instrumented posterior lumbar interbody fusion (PLIF) using unilateral or bilateral polyetheretherketone cages and pedicle screws. METHODS: One hundred eighty-seven cases of degenerative spine that had been followed for at least 18 months were reviewed retrospectively. In 88 cases (147 levels), one cage was inserted, and in 99 cases (152 levels), two cages were inserted. Visual analog scale, Oswestry disability index, and functional rating indices were measured. Lumbar lordosis, lumbar scoliotic and fusion level scoliotic angles, and stable fixation were determined before surgery and 12 months postoperatively on standing x-rays. Amounts of intra- and postoperative blood loss, total quantities transfused, and operation times were also evaluated. RESULTS: No significant differences were found between the two groups in terms of visual analog scale, Oswestry disability index, functional rating indices, lumbar lordosis, lumbar scoliotic angles, fusion level scoliotic angles, or fixation stabilities. However, the amounts of postoperative blood loss, total blood loss, and total transfusion for two-level PLIF using a unilateral cage were statistically smaller than those for two-level PLIF using bilateral cages. Times required for PLIF using a unilateral cage were also significantly shorter than those for PLIF using bilateral cages. CONCLUSION: Unilateral cage and bilateral pedicle screw insertion may be a good alternative surgical option because it provides adequate alignment, balance, and mechanical stability in addition to reducing operative time, blood loss, and transfusion requirements.

Optimal configuration of arthroscopic sliding knots backed up with multiple half-hitches.

The purpose of this biomechanical study was to determine optimized configuration of additional half-hitches placed after various arthroscopic sliding knots. Four commonly used arthroscopic sliding knots, namely, the Duncan loop, the SMC, Weston, and SP knot, were included in this study. All knots were tied with a single-hole knot pusher using a knot-tying simulator. After tying a sliding knot, it was backed up with zero to four additional reverse half-hitches on alternating posts (RHAPs). The first half-hitch was either placed on the same post (conventional RHAP; c-RHAP) or on the loop limb of the sliding knot (switched RHAP; s-RHAP). Ten knots were tied in each configuration using No. 2 Ethibond or No. 1 PDSII sutures. Loop security (loop circumference at 5 N) and knot security (highest load to failure at a cross-head displacement of 3 mm) were measured. Knot configurations, number of RHAPs, or mode of placement of the first half-hitch did not make clinically significant effects on loop security for either suture type. Using No. 2 Ethibond sutures, SMC, SP, and Weston knots required at least three additional half-hitches before knot security plateaued. However, when the first half-hitch was switched to the loop limb of the sliding knot, only two additional half-hitches (2 s-RHAPs) were required to reach maximum knot security. Using No. 1 PDSII sutures, SMC knots needed one additional half-hitch to reach maximum knot security, whereas SP and Weston knots required two half-hitches. However, SMC, SP and Weston knots with switched first half-hitch only required one additional half-hitch (1 s-RHAP) to achieve maximum knot security. This study demonstrated that switching the post just after the sliding knot could save one half-hitch without compromising knot security. That means that sliding knots with 2 s-RHAPs for No. 2 Ethibond and 1 s-RHAPs for No. 1 PDSII achieved knot security comparable to that of sliding knots with 3 c-RHAPs and with 2 c-RHAPs, respectively. The results suggested that the optimized configuration for arthroscopic sliding knots required a secure sliding knot, such as, the SMC, SP, or Weston knots and 2 s-RHAPs when No. 2 Ethibond or No. 1 PDSII suture materials were used considering the unraveling tendency of No. 1 PDSII. Results of the current study may be helpful for establishing arthroscopic sliding knot-tying routines for best results in clinical practice.

Effect of intraoperative position used in posterior lumbar interbody fusion on the maintenance of lumbar lordosis.

OBJECT: The objective in this study was to compare retrospectively the use of different operating tables with different positions for posterior lumbar interbody fusion (PLIF) and the effect on intraoperative and postoperative lumbar lordosis and segmental lordosis. METHODS: One hundred seventy-two patients with degenerative disease of the lumbar spine who underwent posterior decompression and PLIF in which a 0 degrees polyetheretherketone cage and pedicle screw fixation were used were evaluated. Ninety-one patients underwent surgery on a Wilson table (Group I) and 81 patients were treated on an OSI Jackson spinal table (Group II). Preoperative standing, intraoperative prone, and postoperative standing lateral radiographs were obtained in each patient. The total lumbar and segmental lordosis were compared and analyzed according to the position in which the patients were placed for their operation. RESULTS: The intraoperative total lumbar lordosis was significantly decreased compared with the preoperative value. The postoperative total lumbar lordosis was similar, however, to the preoperative values in both groups. In Group I, the intraoperative segmental lordosis of L2-3 and L3-4 was significantly decreased compared with the pre-operative segmental lordosis. In Group II, the intraoperative segmental lordosis of L3-4, L4-5, L5-S1, and L4-S1 was significantly decreased compared with the preoperative segmental lordosis. The postoperative segmental lordosis of L4-5 was significantly decreased and L2-3 was significantly increased compared with the preoperative lordosis in both groups. CONCLUSIONS: Intraoperative position does not affect postoperative total lumbar lordosis and segmental lordosis in short-segment PLIF of the lumbar spine in a retrospective analysis of the surgical procedure to maintain lordosis.