Context
Given the importance of spatial heterogeneity in altering dispersal, interspecific interactions, and population persistence, high rates of habitat homogenisation across the globe are a ...concern. In river ecosystems, confluences likely act as heterogeneity ‘hotspots’ by creating discontinuities in longitudinal processes and influences that are propagated both up and down stream networks.
Objectives
We predicted the layout of abiotic conditions around confluences would influence the presence and configuration of spatial heterogeneity, and strongly influence spatial patterns in fish abundance and evenness.
Methods
Twelve replicate mainstem and tributary stream combinations in Canterbury, New Zealand, were electrofished to evaluate how the configuration of flow disturbance (i.e. flooding) characteristics around a confluence (i.e. stable mainstem and disturbed tributary versus disturbed mainstem and stable tributary) influenced fish communities.
Results
The configuration of abiotic conditions around confluences, and position of a sampled reach with respect to the confluence, significantly interacted to create configuration-specific patterns in fish abundance and evenness. Fish abundances were particularly high in disturbed tributaries juxtaposed with stable mainstems, suggesting certain confluence configurations are disproportionately ecologically important. Evenness scores differed significantly downstream of confluences, depending on configuration, with highest fish evenness in reaches downstream of confluences between a stable and a disturbed stream.
Conclusions
These results reveal strongly context-dependent spatial patterns in communities and demonstrate the role of spatially transferred influence in river systems. Understanding importance of not just the presence of heterogeneity, but its configuration and context-dependence in the landscape will assist in identifying sites of ecological significance for management and conservation purposes.
Arylsulfatases require a maturating enzyme to perform a co- or posttranslational modification to form a catalytically essential formylglycine (FGly) residue. In organisms that live aerobically, ...molecular oxygen is used enzymatically to oxidize cysteine to FGly. Under anaerobic conditions, S -adenosylmethionine (AdoMet) radical chemistry is used. Here we present the structures of an anaerobic sulfatase maturating enzyme (anSME), both with and without peptidyl-substrates, at 1.6–1.8 Å resolution. We find that anSMEs differ from their aerobic counterparts in using backbone-based hydrogen-bonding patterns to interact with their peptidyl-substrates, leading to decreased sequence specificity. These anSME structures from Clostridium perfringens are also the first of an AdoMet radical enzyme that performs dehydrogenase chemistry. Together with accompanying mutagenesis data, a mechanistic proposal is put forth for how AdoMet radical chemistry is coopted to perform a dehydrogenation reaction. In the oxidation of cysteine or serine to FGly by anSME, we identify D277 and an auxiliary 4Fe-4S cluster as the likely acceptor of the final proton and electron, respectively. D277 and both auxiliary clusters are housed in a cysteine-rich C-terminal domain, termed SPASM domain, that contains homology to ∼1,400 other unique AdoMet radical enzymes proposed to use 4Fe-4S clusters to ligate peptidyl-substrates for subsequent modification. In contrast to this proposal, we find that neither auxiliary cluster in anSME bind substrate, and both are fully ligated by cysteine residues. Instead, our structural data suggest that the placement of these auxiliary clusters creates a conduit for electrons to travel from the buried substrate to the protein surface.
Giardia lamblia is a binucleate protistan parasite causing significant diarrheal disease worldwide. An inability to target Cas9 to both nuclei, combined with the lack of nonhomologous end joining and ...markers for positive selection, has stalled the adaptation of CRISPR/Cas9-mediated genetic tools for this widespread parasite. CRISPR interference (CRISPRi) is a modification of the CRISPR/Cas9 system that directs catalytically inactive Cas9 (dCas9) to target loci for stable transcriptional repression. Using a Giardia nuclear localization signal to target dCas9 to both nuclei, we developed efficient and stable CRISPRi-mediated transcriptional repression of exogenous and endogenous genes in Giardia. Specifically, CRISPRi knockdown of kinesin-2a and kinesin-13 causes severe flagellar length defects that mirror defects with morpholino knockdown. Knockdown of the ventral disk MBP protein also causes severe structural defects that are highly prevalent and persist in the population more than 5 d longer than defects associated with transient morpholino-based knockdown. By expressing two guide RNAs in tandem to simultaneously knock down kinesin-13 and MBP, we created a stable dual knockdown strain with both flagellar length and disk defects. The efficiency and simplicity of CRISPRi in polyploid Giardia allows rapid evaluation of knockdown phenotypes and highlights the utility of CRISPRi for emerging model systems.
Many Proteobacteria use N -acyl-homoserine lactone (acyl-HSL) quorum sensing to control specific genes. Acyl-HSL synthesis requires unique enzymes that use S -adenosyl methionine as an acyl acceptor ...and amino acid donor. We developed and executed an enzyme-coupled high-throughput cell-free screen to discover acyl-HSL synthase inhibitors. The three strongest inhibitors were equally active against two different acyl-HSL synthases: Burkholderia mallei BmaI1 and Yersinia pestis YspI. Two of these inhibitors showed activity in whole cells. The most potent compound behaves as a noncompetitive inhibitor with a K ᵢ of 0.7 µM and showed activity in a cell-based assay. Quorum-sensing signal synthesis inhibitors will be useful in attempts to understand acyl-HSL synthase catalysis and as a tool in studies of quorum-sensing control of gene expression. Because acyl-HSL quorum-sensing controls virulence of some bacterial pathogens, anti–quorum-sensing chemicals have been sought as potential therapeutic agents. Our screen and identification of acyl-HSL synthase inhibitors serve as a basis for efforts to target quorum-sensing signal synthesis as an antivirulence approach.
Despite their broad anti-infective utility, the biosynthesis of the paradigm carbapenem antibiotic, thienamycin, remains largely unknown. Apart from the first two steps shared with a simple ...carbapenem, the pathway sharply diverges to the more structurally complex members of this class of β-lactam antibiotics, such as thienamycin. Existing evidence points to three putative cobalamin-dependent radical S-adenosylmethionine (RS) enzymes, ThnK, ThnL, and ThnP, as potentially being responsible for assembly of the ethyl side chain at C6, bridgehead epimerization at C5, installation of the C2-thioether side chain, and C2/3 desaturation. The C2 substituent has been demonstrated to be derived by stepwise truncation of CoA, but the timing of these events with respect to C2-S bond formation is not known. We show that ThnK of the three apparent cobalamin-dependent RS enzymes performs sequential methylations to build out the C6-ethyl side chain in a stereocontrolled manner. This enzymatic reaction was found to produce expected RS methylase coproducts S-adenosylhomocysteine and 5'-deoxyadenosine, and to require cobalamin. For double methylation to occur, the carbapenam substrate must bear a CoA-derived C2-thioether side chain, implying the activity of a previous sulfur insertion by an as-yet unidentified enzyme. These insights allow refinement of the central steps in complex carbapenem biosynthesis.
The radical S-adenosyl-L-methionine (SAM) enzymes RlmN and Cfr methylate 235 ribosomal RNA, modifying the C2 or C8 position of adenosine 2503. The methyl groups are installed by a two-step sequence ...involving initial methylation of a conserved Cys residue (RlmN Cys 355 ) by SAM. Methyl transfer to the substrate requires reductive cleavage of a second equivalent of SAM. Crystal structures of RlmN and RlmN with SAM show that a single molecule of SAM coordinates the 4Fe-4S cluster. Residue Cys 355 is S-methylated and located proximal to the SAM methyl group, suggesting the SAM that is involved in the initial methyl transfer binds at the same site. Thus, RlmN accomplishes its complex reaction with structural economy, harnessing the two most important reactivities of SAM within a single site.
Purpose
Anterior lumbar surgery is a common procedure for anterior lumbar interbody fusion (ALIF) and artificial disc replacement (ADR). Our aim was to study the exposure related complications for ...anterior lumbar spinal surgery performed by spinal surgeons.
Methods
A retrospective review was performed for 304 consecutive patients who underwent anterior lumbar spinal surgery over 10 years (2001–2010) at our institution. Each patient’s records were reviewed for patients’ demographics, diagnosis, level(s) of surgery, procedure and complications related to access surgery. Patients undergoing anterior lumbar access for tumour resection, infection, trauma and revision surgeries were excluded.
Results
All patients underwent an anterior paramedian retroperitoneal approach from the left side. The mean age of patients was 43 years (10–73; 197 males, 107 females). Indications for surgery were degenerative disc disease (DDD 255), degenerative spondylolisthesis (23), scoliosis (18), iatrogenic spondylolisthesis (5) and pseudoarthrosis (3). The procedures performed were single level surgery—L5/S1 (
n
= 147), L4/5 (
n
= 62), L3/4 (
n
= 7); two levels—L4/5 and L5/S1 (
n
= 74), L3/4 and L4/5 (
n
= 4); three levels—L3/4, L4/5, L5/S1 (
n
= 5); four levels—L2/3, L3/4, L4/5, L5/S1 (
n
= 5). The operative procedures were single level ADR (
n
= 131), a single level ALIF (
n
=
87) with or without posterior fusion, two levels ALIF (
n
= 54), two levels ADR (
n
= 14), a combination of ADR/ALIF (
n
= 10), three levels ALIF (
n
= 1), three levels ADR/ALIF/ALIF (
n
= 1), ADR/ADR/ALIF (
n
= 2), four levels ALIF (
n
= 1) and finally 3 patients underwent a four level ADR/ADR/ALIF/ALIF. The overall complication rate was 61/304 (20 %). This included major complications (6.2 %)—venous injury requiring suture repair (
n
= 14, 4.6 %) and arterial injury (
n
= 5 1.6 %, 3 repaired, 2 thrombolysed). Minor complications (13.8 %) included venous injury managed without repair (
n
= 5, 1.6 %), infection (
n
= 13, 4.3 %), incidental peritoneal opening (
n
= 12, 3.9 %), leg oedema (
n
= 2, 0.6 %) and others (
n
= 10, 3.3 %). We had no cases of retrograde ejaculation.
Conclusion
We report a very thorough and critical review of our anterior lumbar access surgeries performed mostly for DDD and spondylolisthesis at L4/5 and L5/S1 levels. Vascular problems of any type (24/304, 7.8 %) were the most common complication during this approach. The incidence of major venous injury requiring repair was 14/304 (4.6 %) and arterial injury 5/304 (1.6 %). The requirement for a vascular surgeon with the vascular injury was 9/304 (3 %; 5 arterial injuries; 4 venous injuries). This also suggests that the majority of the major venous injuries were repaired by the spinal surgeon (10/14, 71 %). Our results are comparable to other studies and support the notion that anterior access surgery to the lumbar spine can be performed safely by spinal surgeons. With adequate training, spinal surgeons are capable of performing this approach without direct vascular support, but they should be available if required.
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