Perioperative Protocol of Ankle Fracture and Distal Radius Fracture Based on Enhanced Recovery after Surgery Program: A Multicenter Prospective Clinical Controlled study.

Background: The enhanced recovery after surgery (ERAS) program is aimed to shorten patients' recovery process and improve clinical outcomes. This study aimed to compare the outcomes between the ERAS program and the traditional pathway among patients with ankle fracture and distal radius fracture. Methods: This is a multicenter prospective clinical controlled study consisting of 323 consecutive adults with ankle fracture from 12 centers and 323 consecutive adults with distal radial fracture from 13 centers scheduled for open reduction and internal fixation between January 2017 and December 2018. According to the perioperative protocol, patients were divided into two groups: the ERAS group and the traditional group. The primary outcome was the patients' satisfaction of the whole treatment on discharge and at 6 months postoperatively. The secondary outcomes include delapsed time between admission and surgery, length of hospital stay, postoperative complications, functional score, and the MOS item short form health survey-36. Results: Data describing 772 patients with ankle fracture and 658 patients with distal radius fracture were collected, of which 323 patients with ankle fracture and 323 patients with distal radial fracture were included for analysis. The patients in the ERAS group showed higher satisfaction levels on discharge and at 6 months postoperatively than in the traditional group (P < 0.001). In the subgroup analysis, patients with distal radial fracture in the ERAS group were more satisfied with the treatment (P=0.001). Furthermore, patients with ankle fracture had less time in bed (P < 0.001) and shorter hospital stay (P < 0.001) and patients with distal radial fracture received surgery quickly after being admitted into the ward in the ERAS group than in the traditional group (P=0.001). Conclusions: Perioperative protocol based on the ERAS program was associated with high satisfaction levels, less time in bed, and short hospital stay without increased complication rate and decreased functional outcomes.

Sec15 links bud site selection to polarised cell growth and exocytosis in Candida albicans.

The exocyst plays a crucial role in the targeting of secretory vesicles to the plasma membrane during exocytosis. It has been shown to be involved in diverse cellular processes including yeast budding. However, the mechanism of the exocyst regulating yeast budding has not been fully elucidated. Here we report a novel interaction between the exocyst component Sec15 and the Ras-family GTPase Rsr1, a master regulator of bud-site-selection system, in the fungus Candida albicans. We present several lines of evidence indicating physical and genetic interaction of Sec15 with Rsr1. In vitro binding assays and co-immunoprecipitation studies showed that Sec15 associated physically with Rsr1. Deletion of RSR1 completely abolished the polarised localisation of Sec15 as well as all the other exocyst components in both yeast and hyphal cells, suggesting a functional interaction between Sec15 and Rsr1. We also show that C. albicans Sec15 interacts directly with the polarity determinant Bem1 and the type V myosin, Myo2. Disruption of the interaction by shutting off SEC15 results in mislocaliztion of Bem1-GFP. These findings highlight the important role of Sec15 in polarised cell growth by providing a direct functional link between bud-site-selection and exocytosis.

CDK regulates septin organization through cell-cycle-dependent phosphorylation of the Nim1-related kinase Gin4.

Cyclin-dependent kinases (CDKs) regulate septin organization in a cell-cycle-dependent manner in yeast. However, the mechanism remains unclear. Here, we show that the Candida albicans CDK Cdc28 phosphorylates the Nim1-related kinase Gin4, a known septin regulator, activating its kinase activity, which in turn phosphorylates the Sep7 septin. Gin4 contains a cluster of CDK phosphorylation sites near the kinase domain. Replacing serine/threonine with alanine in these sites prevents Gin4 activation, weakens its association with Sep7, alters Sep7 dynamics and causes morphological and cytokinetic defects. By contrast, phosphomimetic mutation enhances the kinase activity with only moderate deteriorating effects. We also found that Gin4 has both kinase-independent and -dependent functions, acting during G1 phase and mitosis, respectively, with the former being essential for septin ring assembly. Thus, we have identified a previously unknown signaling pathway linking CDKs and the septins that provides new insights into the mechanisms controlling septin organization and function in coordination with cell-cycle phases.

CDK-dependent phosphorylation of Mob2 is essential for hyphal development in Candida albicans.

Nuclear Dbf2-related (NDR) protein kinases are essential components of regulatory pathways involved in cell morphogenesis, cell cycle control, and viability in eukaryotic cells. For their activity and function, these kinases require interaction with Mob proteins. However, little is known about how the Mob proteins are regulated. In Candida albicans, the cyclin-dependent kinase (CDK) Cdc28 and the NDR kinase Cbk1 are required for hyphal growth. Here we demonstrate that Mob2, the Cbk1 activator, undergoes a Cdc28-dependent differential phosphorylation on hyphal induction. Mutations in the four CDK consensus sites in Mob2 to Ala significantly impaired hyphal development. The mutant cells produced short hyphae with enlarged tips that displayed an illicit activation of cell separation. We also show that Cdc28 phosphorylation of Mob2 is essential for the maintenance of polarisome components at hyphal tips but not at bud tips during yeast growth. Thus we have found a novel signaling pathway by which Cdc28 controls Cbk1 through the regulatory phosphorylation of Mob2, which is crucial for normal hyphal development.

The IQGAP Iqg1 is a regulatory target of CDK for cytokinesis in Candida albicans.

Cyclin-dependent kinases (CDKs) drive and coordinate multiple cell-cycle events, including construction and contraction of the actomyosin ring during cytokinesis. However, it remains unclear whether CDKs regulate cytokinesis by directly targeting components of the ring. In a search for proteins containing consensus CDK phosphorylation sites in Candida albicans, we found that the IQGAP Iqg1 contains two dense clusters of 19 such sites flanking the actin-interacting CH domain. Here, we show that Iqg1 is indeed a phosphoprotein that undergoes cell-cycle-dependent phosphorylation and can be phosphorylated by purified Clb-Cdc28 kinases in vitro. Mass spectrometry identified several phosphoserine and phosphothreonine residues among these CDK sites. Mutating 15 of the CDK phosphorylation sites with alanine markedly reduced Iqg1 phosphorylation in vivo. The 15A mutation greatly stabilized Iqg1, caused both premature assembly and delayed disassembly of the actomyosin ring, blocked Iqg1 interaction with the actin-nucleating proteins Bni1 and Bnr1, and resulted in defects in cytokinesis. Our data therefore strongly support the idea that the Cdc28 CDK regulates cytokinesis partly by directly phosphorylating the actomyosin ring component Iqg1.

Cyclin-dependent kinases control septin phosphorylation in Candida albicans hyphal development.

Cyclin-dependent kinases (Cdks) control cytoskeleton polarization in yeast morphogenesis. However, the target and mechanism remain unclear. Here, we show that the Candida albicans Cdk Cdc28, through temporally controlled association with two cyclins Ccn1 and Hgc1, rapidly establishes and persistently maintains phosphorylation of the septin cytoskeleton protein Cdc11 for hyphal development. Upon hyphal induction, Cdc28-Ccn1 binds to septin complexes and phosphorylates Cdc11 on Ser394, a nonconsensus Cdk target. This phosphorylation requires prior phosphorylation on Ser395 by the septin-associated kinase Gin4. Mutating Ser394 or Ser395 blocked Cdc11 phosphorylation on Ser394 and impaired hyphal morphogenesis. Reconstitution experiments using purified Cdc28-Ccn1, Gin4, and septins reproduced phosphorylations on the same residues. Transient septin-Cdc28 associations were also detected prior to bud and mating-projection emergence in S. cerevisiae. Our study uncovers a direct link between the cell-cycle engine and the septin cytoskeleton that may be part of a conserved mechanism underlying polarized morphogenesis.

Candida albicans hyphal morphogenesis occurs in Sec3p-independent and Sec3p-dependent phases separated by septin ring formation.

The growing tips of Candida albicans hyphae are sites of polarized exocytosis. Mammalian septins have been implicated in regulating exocytosis and C. albicans septins are known to localize at hyphal tips, although their function here is unknown. Here, we report that C. albicans cells deleted of the exocyst subunit gene SEC3 can grow normal germ tubes, but are unable to maintain tip growth after assembly of the first septin ring, resulting in isotropic expansion of the tip. Deleting either of the septin genes CDC10 or CDC11 caused Sec3p mislocalization and surprisingly, also restored hyphal development in the sec3Delta mutant without rescuing the temperature sensitivity. Co-immunoprecipitation experiments detected association of the septin Cdc3p with the exocyst subunits Sec3p and Sec5p. Our results reveal that C. albicans hyphal development occurs through Sec3p-independent and dependent phases, and provide strong genetic and biochemical evidence for a role of septins in polarized exocytosis.

The formin family protein CaBni1p has a role in cell polarity control during both yeast and hyphal growth in Candida albicans.

Formins are conserved eukaryotic proteins playing key roles in regulating cell polarity. We have characterized the roles of a formin CaBni1p in the polymorphic fungus Candida albicans. CaBni1p localized persistently to hyphal tips during hyphal growth but to distinct growth sites at different cell cycle stages during yeast growth. Cabni1Delta yeast cells exhibited several morphological defects, such as round and enlarged cells, widened bud necks and a random budding pattern. Although Cabni1Delta cells could still undergo yeast-hypha growth switch, the hyphae were markedly swollen. Cabni1Delta also showed defects in spindle and cytoplasmic microtubule orientation and positioning. Coincidentally, the spindle orientation protein CaKar9p in Cabni1Delta yeast cells appeared as multiple random cortical spots, in contrast to the single spot at the bud tip of many wild-type cells. Interestingly, several defects manifested in Cabni1Delta yeast cells were partially corrected during hyphal growth. We found that the second formin CaBnr1p was recruited to hyphal tips, while it localized only to the bud neck during yeast growth. This behavior of CaBnr1p may play a key role in correcting Cabni1Delta defects during hyphal growth. Cabni1Delta exhibited reduced virulence in mice. These results indicate that the formins play an important role in Candida albicans polarized growth and CaBni1p's function is required for virulence.

Cloning, characterization and tissue specific expression of a sucrose synthase gene from tropical epiphytic CAM orchid Mokara Yellow.

A full-length cDNA encoding sucrose synthase was isolated from the tropical epiphytic CAM orchid Mokara Yellow. The cDNA is 2748bp in length containing an open reading frame of 2447bp encoding 816 amino acids with a predicted molecular mass of 93.1 kDa. The deduced amino acid sequence of M. Yellow sucrose synthase (Msus1) shares more than 80% identity with those from other monocotyledonous plants. The sucrose synthase gene was demonstrated to encode a functional sucrose synthase protein by expression as recombinant protein in Escherichia coli. Northern blot analysis showed that the expression pattern of Msus1 mRNA is tissue specific with highest levels in strong sinks such as expanding leaves and root tips, but not detectable in mature leaves and flowers. Incubation with sugars resulted in a significant increase in the steady-state Msus1 mRNA levels in shoots of seedlings.

Cloning of a sucrose-phosphate synthase gene highly expressed in flowers from the tropical epiphytic orchid Oncidium Goldiana.

Sucrose-phosphate synthase (SPS) is one of the key regulatory enzymes in carbon assimilation and partitioning in plants. It plays a crucial role in the production of sucrose in photosynthetic cells. The cloning and expression analysis of a full-length cDNA encoding SPS from tropical epiphytic orchid hybrid Oncidium Goldiana are reported here. The cDNA designated as sps1 is 3820 bp in length with an open reading frame of 3183 bp encoding 1061 amino acids. The deduced amino acid sequence of O. Goldiana sps1 shows 56% and 69% homology with those of maize SPS and spinach SPS, respectively. The high level expression of O. Goldiana sps1 in the flower suggests that it might play an important role in flowering. Growth under higher irradiance and elevated CO2 leads to an accumulation of the sps1 transcript in the photosynthetic leaves. It appears that SPS gene expression in photosynthetic leaves is associated with the leaf photosynthetic rate.