Hoffa fractures are associated with concomitant soft tissue injures and a high postoperative complication rate.

INTRODUCTION: Hoffa fractures are a rare and often overlooked entity. The main goal of surgical treatment is to restore the articular surface and maintain knee function. However, current clinical data indicate heterogeneous outcomes. The aim of this multicenter study was to obtain a representative data set of patients with isolated Hoffa fractures with special emphasis on concomitant soft tissue injuries, diagnostic algorithms, treatment strategies and functional outcomes. MATERIALS AND METHODS: Participating Level I trauma centres were asked to review their internal database for isolated Hoffa fractures treated surgically between 2010 and 2020. Demographics, mechanism of injury, diagnostic and therapeutic algorithm, Letenneur classification, concomitant soft tissue injuries, and postoperative knee function and complications were analysed. RESULTS: A total of 56 patients from six participating trauma centres were included. The median age at injury was 45 years (15-94) with a median follow-up of 19 months (2-108). The most common mechanism of injury was high-energy trauma, with unicondylar lateral Letenneur type I and II fractures being the most common. Surgical treatment was independent of the type of fracture and included isolated screw fixation, combined plate and screw fixation and isolated plate osteosynthesis. Isolated screw fixation resulted in significantly better range of motion (ROM) values (p = 0.032), but the highest number of postoperative complications (n = 14/20, n.s.) compared to the other fixation techniques. The highest number of fixation failures requiring revision was observed in the plate and screw fixation group (n = 3/8, p = 0.008). Osteochondral flake fractures (n = 12/43, 27%) and lateral meniscus injuries (n = 5/49, 10%) were commonly seen in Hoffa fractures. CONCLUSIONS: Treatment of Hoffa fractures with screw fixation resulted in significantly better functional outcomes, probably due to less comminuted fractures. Concomitant cartilage, meniscal and ligamentous injuries are common and warrant preoperative recognition and management.

Current standard of care for distal femur fractures in Germany and Switzerland.

BACKGROUND: Distal femur fractures occur with an incidence of 4.5/100,000 and show a prevalence of 0.4%. Causes include low-impact trauma in older patients and high-impact trauma in younger patients without pre-existing medical conditions. The aim of this study was to perform a comprehensive evaluation of trauma mechanisms, trauma-promoting factors, comorbidities, medication history and type of surgical care to provide an overview of the causes of injury and the most appropriate therapeutic approach. METHODS: In this multicenter cohort study a retrospective analysis of 229 patients who sustained a distal femur fracture between January 2011 and December 2020 was performed. Individual fracture patterns, fracture predisposing factors, concomitant disease profiles, medication history, treatment strategy and associated complications were analyzed. RESULTS: 229 patients were included in the retrospective analysis. A total of 113-type 33 A, 50-type 33 B and 66-type 33 C fractures were diagnosed, of whom 92% received a lateral locking plate osteosynthesis. There was a complication in 14.4% of all cases, of which 6.1% were attributable to infection. Significant risk factors for developing a complication were an increased BMI (29.9 ± 8.5 kg/m2; p = 0.04), fracture displacement of over half a shaft width (p < 0.001) and AOC fractures (p < 0,016), specifically C2 fractures (p < 0,008). CONCLUSION: In this multicenter retrospective cohort study, lateral locking plate osteosynthesis was the method of choice and was selected in over 90% of cases, regardless of the fracture classification and risk factors. A complication rate of 14.4% emphasizes the necessary analysis of patient- and care-specific risk factors and a resulting adjustment of the therapy strategy. An increased BMI (29.9 ± 8.5 kg/m2; p = 0.04), fracture displacement of over half a shaft width (p < 0.001) and AOC fractures (p < 0,016), specifically C2 fractures (p < 0,008) increase the risk of developing a complication and should prompt an early switch to a treatment strategy that provides more stability.

[Osteosynthesis of extra-articular proximal tibial fractures]. / Osteosynthese bei extraartikulärer proximaler Tibiafraktur.

The treatment of extra-articular proximal tibial fractures is a therapeutic challenge due to the frequently significant soft tissue injury, the effect of the deforming forces and the need for an exact restoration of the bony alignment. Various methods of osteosynthesis are available for surgical stabilization. The locking plate osteosynthesis is the most frequently used procedure because of its good biomechanical stability, especially in osteoporotic bones, and the protection of the periosteal blood flow. Depending on the extent and stability of the defect zone, especially in the case of a medial comminuted zone and the bone quality, bilateral plate osteosynthesis can be necessary. If the proximal fragment is big enough, closed reduction and intramedullary nailing are possible. In the case of severely compromised soft tissue or very short epiphyseal fragments, the construction of an external fixator, e.g. hybrid external fixator, is recommended, which also allows definitive treatment under early full weight bearing. The most important complications are axial and torsional malalignments.

Structure, function and biosynthesis of the Mycobacterium tuberculosis cell wall: arabinogalactan and lipoarabinomannan assembly with a view to discovering new drug targets.

In spite of effective antibiotics to treat TB (tuberculosis) since the early 1960s, we enter the new millennium with TB, currently the leading cause of death from a single infectious agent, killing more than three million people worldwide each year. Thus an understanding of drug-resistance mechanisms, the immunobiology of cell wall components to elucidate host-pathogen interactions and the discovery of new drug targets are now required for the treatment of TB. Above the plasma membrane is a classical chemotype IV PG (peptidoglycan) to which is attached the macromolecular structure, mycolyl-arabinogalactan, via a unique diglycosylphosphoryl bridge. This review will discuss the assembly of the mAGP (mycolyl-arabinogalactan-peptidoglycan), its associated glycolipids and the site of action of EMB (ethambutol), bringing forward a new era in TB research and focus on new drugs to combat multidrug resistant TB.

3-Phosphoglycerate dehydrogenase from Corynebacterium glutamicum: the C-terminal domain is not essential for activity but is required for inhibition by L-serine.

The serA gene of Corynebacterium glutamicum coding for 3-phosphoglycerate dehydrogenase (PGDH) was isolated and functionally characterized. It encodes a polypeptide of 530 aminoacyl residues (aa), which is substantially longer than the corresponding Escherichia coli polypeptide of 410 aa. The difference is largely due to an additional stretch of aa in the carboxy- (C)-terminal part of the polypeptide. Overexpression of serA in C. glutamicum results in a 16-fold increase in specific PGDH activity to 2.1 U/mg protein, with activity being inhibited by high concentrations of L-serine. A set of muteins that were progressively truncated at the C-terminal end was constructed. When overexpressed, mutein SerADelta197 showed a specific PGDH dehydrogenase activity of 1.3 U/mg protein, with the activity no longer being sensitive to L-serine. Gel filtration experiments showed that wild type PGDH is a homotetramer, whereas mutein SerADelta197 constitutes a dimer. Thus, the specific regulatory features of C. glutamicum PGDH are due to the C-terminal part of the polypeptide, which can be deleted with almost no effect on the catalytic activity of the enzyme.

Influence of threonine exporters on threonine production in Escherichia coli.

Threonine production in Escherichia coli threonine producer strains is enhanced by overexpression of the E. coli rhtB and rhtC genes or by heterologous overexpression of the gene encoding the Corynebacterium glutamicum threonine excretion carrier, thrE. Both E. coli genes give rise to a threonine-resistant phenotype when overexpressed, and they decrease the accumulation of radioactive metabolites derived from [(14)C] L-threonine. The evidence presented supports the conclusion that both RhtB and RhtC catalyze efflux of L-threonine and other structurally related neutral amino acids, but that the specificities of these two carriers differ substantially.

Expression of genes of lipid synthesis and altered lipid composition modulates L-glutamate efflux of Corynebacterium glutamicum.

L-Glutamate is made with Corynebacterium glutamicum on a scale of more than 106 tons/year. Nevertheless, formation of this amino acid is enigmatic and there is very limited molecular information available to unravel the apparently complex conditions leading to L-glutamate efflux. Here, we report the isolation and overexpression of the genes involved in lipid synthesis: acp, fadD 15, cma, cls, pgsA2, cdsA, gpsA, and plsC, and the inactivation of cma and cls. In addition, the consequences for phospholipid content, temperature sensitivity, as well as detergent-independent and detergent-dependent L-glutamate efflux were quantified. An in part strong alteration of the phospholipid composition was achieved; for instance, overexpression offadD15 encoding an acyl-CoA ligase resulted in an increase of phosphatidyl inositol from 12.6 to 30.2%. All strains, except that overexpressing acp (acyl carrier protein), exhibited increased temperature sensitivity, with the strongest sensitivity present upon cls (cardiolipin synthetase) inactivation. As a consequence of the genetically modified lipid synthesis, L-glutamate efflux changed quite dramatically; for instance, overexpression of plsC (acylglycerolacyl transferase) resulted in a detergent-triggered increase of L-glutamate accumulation from 92 mM to 108 mM, whereas acp overexpression reduced the accumulation to 24 mM. With some of the overexpressed genes, substantial L-glutamate excretion even without detergent addition was obtained when the fermentation temperature was elevated. These data show that the chemical and physical properties of the cytoplasmic membrane are altered and suggest that this is a necessary precondition to achieve L-glutamate efflux.

L-threonine export: use of peptides to identify a new translocator from Corynebacterium glutamicum.

Bacterial mechanisms for the uptake of peptides and their hydrolysis to amino acids are known in great detail, whereas much less is known about the fates of the peptide-derived amino acids. We show that the addition of L-threonine-containing di- or tripeptides results in reduction of the growth of Corynebacterium glutamicum, with concomitant high intracellular accumulation of L-threonine to up to 130 mM. Using transposon mutagenesis and isolation of mutants with increased Thr peptide sensitivity, nine open reading frames (ORFs) were identified, almost all encoding hypothetical proteins of unknown function. Three ORFs encode membrane proteins. Their individual functional characterizations in the wild-type background led to the identification of thrE. Upon thrE overexpression, growth is no longer sensitive to the presence of the Thr peptide, and L-threonine is exported at a rate of 3.8 nmol min(-1) mg of dry weight(-1), whereas the rate of export of a thrE inactivation mutant is reduced to 1.1 nmol min(-1) mg of dry weight(-1). In addition to L-threonine, L-serine is also a substrate for the exporter. The exporter exhibits nine predicted transmembrane-spanning helices with long charged C and N termini and with an amphipathic helix present within the N terminus. All these data suggest that the carrier encoded by thrE serves to export small molecules such as L-threonine and that the carrier is a prototype of a new translocator family. Homologues of ThrE are present in Mycobacterium tuberculosis and Streptomyces coelicolor.

Expression control and specificity of the basic amino acid exporter LysE of Corynebacterium glutamicum.

LysE of Corynebacterium glutamicum belongs to a large new superfamily of translocators whose members are probably all involved in the export of small solutes. Here, the transcript initiation site of lysE, and its divergently transcribed regulator gene, lysG, are identified. Single-copy transcriptional fusions of lysE with lacZ, and titration experiments, show that LysG is the positive regulator of lysE expression enabling its up to 20-fold induction. This induction requires the presence of a coinducer, which is either intracellular L-lysine, or L-arginine. A competition experiment showed that LysE exports these two basic amino acids at comparable rates of about 0.75 nmol min(-1) (mg dry wt)(-1). Although L-histidine and L-citrulline also act as coinducers of lysE expression, these two amino acids are not exported by LysE. As is evident from the analysis of a lysEG deletion mutant, the physiological role of the lysEG system is to prevent bacteriostasis due to elevated L-lysine or L-arginine concentrations that arise during growth in the presence of peptides or in mutants possessing a deregulated biosynthesis pathway. C. glutamicum has additional export activities other than those of LysE for exporting L-histidine, L-citrulline and L-ornithine.

Metabolic engineering for L-lysine production by Corynebacterium glutamicum.

Corynebacterium glutamicum has been used since several decades for the large-scale production of amino acids, esp. L-glutamate and L-lysine. After initial successes of random mutagenesis and screening approaches, further strain improvements now require a much more rational design, i.e. metabolic engineering. Not only recombinant DNA technology but also mathematical modelling of metabolism as well as metabolic flux analysis represent important metabolic engineering tools. This review covers as state-of-the-art examples of these techniques the genetic engineering of the L-lysine biosynthetic pathway resulting in a vectorless strain with significantly increased dihydrodipicolinate synthase activity, and the detailed metabolic flux analysis by 13C isotopomer labelling strategies of the anaplerotic enzyme activities in C. glutamicum resulting in the identification of gluconeogenic phosphoenolpyruvate carboxykinase as a limiting enzyme.

Pathway analysis and metabolic engineering in Corynebacterium glutamicum.

The gram-positive bacterium Corynebacterium glutamicum is used for the industrial production of amino acids, e.g. of L-glutamate and L-lysine. During the last 15 years, genetic engineering and amplification of genes have become fascinating methods for studying metabolic pathways in greater detail and for the construction of strains with the desired genotypes. In order to obtain a better understanding of the central metabolism and to quantify the in vivo fluxes in C. glutamicum, the [13C]-labelling technique was combined with metabolite balancing to achieve a unifying comprehensive pathway analysis. These methods can determine the flux distribution at the branch point between glycolysis and the pentose phosphate pathway. The in vivo fluxes in the oxidative part of the pentose phosphate pathway calculated on the basis of intracellular metabolite concentrations and the kinetic constants of the purified glucose-6-phosphate and 6-phosphogluconate dehydrogenases determined in vitro were in full accordance with the fluxes measured by the [13C]-labelling technique. These data indicate that the oxidative pentose phosphate pathway in C. glutamicum is mainly regulated by the ratio of NADPH/NADP concentrations and the specific activity of glucose-6-phosphate dehydrogenase. The carbon flux via the oxidative pentose phosphate pathway correlated with the NADPH demand for L-lysine synthesis. Although it has generally been accepted that phosphoenolpyruvate carboxylase fulfills a main anaplerotic function in C. glutamicum, we recently detected that a biotin-dependent pyruvate carboxylase exists as a further anaplerotic enzyme in this bacterium. In addition to the activities of these two carboxylases three enzymes catalysing the decarboxylation of the C4 metabolites oxaloacetate or malate are also present in this bacterium. The individual flux rates at this complex anaplerotic node were investigated by using [13C]-labelled substrates. The results indicate that both carboxylation and decarboxylation occur simultaneously in C. glutamicum so that a high cyclic flux of oxaloacetate via phosphoenolpyruvate to pyruvate was found. Furthermore, we detected that in C. glutamicum two biosynthetic pathways exist for the synthesis of DL-diaminopimelate and L-lysine. As shown by NMR spectroscopy the relative use of both pathways in vivo is dependent on the ammonium concentration in the culture medium. Mutants defective in one pathway are still able to synthesise enough L-lysine for growth, but the L-lysine yields with overproducers were reduced. The luxury of having these two pathways gives C. glutamicum an increased flexibility in response to changing environmental conditions and is also related to the essential need for DL-diaminopimelate as a building block for the synthesis of the murein sacculus.

In vivo quantification of parallel and bidirectional fluxes in the anaplerosis of Corynebacterium glutamicum.

The C(3)-C(4) metabolite interconversion at the anaplerotic node in many microorganisms involves a complex set of reactions. C(3) carboxylation to oxaloacetate can originate from phosphoenolpyruvate and pyruvate, and at the same time multiple C(4)-decarboxylating enzymes may be present. The functions of such parallel reactions are not yet fully understood. Using a (13)C NMR-based strategy, we here quantify the individual fluxes at the anaplerotic node of Corynebacterium glutamicum, which is an example of a bacterium possessing multiple carboxylation and decarboxylation reactions. C. glutamicum was grown with a (13)C-labeled glucose isotopomer mixture as the main carbon source and (13)C-labeled lactate as a cosubstrate. 58 isotopomers as well as 15 positional labels of biomass compounds were quantified. Applying a generally applicable mathematical model to include metabolite mass and carbon labeling balances, it is shown that pyruvate carboxylase contributed 91 +/- 7% to C(3) carboxylation. The total in vivo carboxylation rate of 1.28 +/- 0.14 mmol/g dry weight/h exceeds the demand of carboxylated metabolites for biosyntheses 3-fold. Excess oxaloacetate was recycled to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase. This shows that the reactions at the anaplerotic node might serve additional purposes other than only providing C(4) metabolites for biosynthesis.

Characterization of a bordetella pertussis diaminopimelate (DAP) biosynthesis locus identifies dapC, a novel gene coding for an N-succinyl-L,L-DAP aminotransferase.

The functional complementation of two Escherichia coli strains defective in the succinylase pathway of meso-diaminopimelate (meso-DAP) biosynthesis with a Bordetella pertussis gene library resulted in the isolation of a putative dap operon containing three open reading frames (ORFs). In line with the successful complementation of the E. coli dapD and dapE mutants, the deduced amino acid sequences of two ORFs revealed significant sequence similarities with the DapD and DapE proteins of E. coli and many other bacteria which exhibit tetrahydrodipicolinate succinylase and N-succinyl-L,L-DAP desuccinylase activity, respectively. The first ORF within the operon showed significant sequence similarities with transaminases and contains the characteristic pyridoxal-5'-phosphate binding motif. Enzymatic studies revealed that this ORF encodes a protein with N-succinyl-L,L-DAP aminotransferase activity converting N-succinyl-2-amino-6-ketopimelate, the product of the succinylase DapD, to N-succinyl-L,L-DAP, the substrate of the desuccinylase DapE. Therefore, this gene appears to encode the DapC protein of B. pertussis. Apart from the pyridoxal-5'-phosphate binding motif, the DapC protein does not show further amino acid sequence similarities with the only other known enzyme with N-succinyl-L,L-DAP aminotransferase activity, ArgD of E. coli.

Attenuation control of ilvBNC in Corynebacterium glutamicum: evidence of leader peptide formation without the presence of a ribosome binding site.

The ilvBNC operon of Corynebacterium glutamicum encodes acetohydroxy acid synthase and isomero-reductase, which are key enzymes of L-isoleucine, L-valine and L-leucine syntheses. In this study we identified the transcript initiation site of ilvBNC operon 292 nucleotides in front of the first structural gene, and detected the formation of a short transcript from the leader region in addition to the full-length transcript of the operon. This identifies the control of ilvBNC transcription by an attenuation mechanism involving antitermination. Mutations in the leader region were made and their effect on the operon expression in ilvB'lacZ fusions was quantified. Although a presumed leader-peptide-coding region is only one nucleotide away from the transcript initiation site determined, there is clear evidence to support the formation of this leader peptide: (i) the substitution of initiation codon ATG of the peptide by AGG reduced lacZ expression of the appropriate fusion construct to 19%; (ii) the replacement of three subsequent Val codons by Ala codons resulted in the loss of Val-dependent expression; and (iii) a leader peptide LacZ fusion resulted in active beta-galactosidase. Based on these results, it is concluded that transcription of ilvBNC is controlled by a translational-coupled attenuation mechanism. The absence of a ribosome binding site for leader peptide formation means that additional mechanisms may contribute to the transcription control at the decoding initiation step in the leader peptide formation.

D-Pantothenate synthesis in Corynebacterium glutamicum and use of panBC and genes encoding L-valine synthesis for D-pantothenate overproduction.

D-Pantothenate is synthesized via four enzymes from ketoisovalerate, which is an intermediate of branched-chain amino acid synthesis. We quantified three of these enzyme activities in Corynebacterium glutamicum and determined specific activities ranging from 0.00014 to 0.001 micromol/min mg (protein)-1. The genes encoding the ketopantoatehydroxymethyl transferase and the pantothenate synthetase were cloned, sequenced, and functionally characterized. These studies suggest that panBC constitutes an operon. By using panC, an assay system was developed to quantify D-pantothenate. The wild type of C. glutamicum was found to accumulate 9 micrograms of this vitamin per liter. A strain was constructed (i) to abolish L-isoleucine synthesis, (ii) to result in increased ketoisovalerate formation, and (iii) to enable its further conversion to D-pantothenate. The best resulting strain has ilvA deleted from its chromosome and has two plasmids to overexpress genes of ketoisovalerate (ilvBNCD) and D-pantothenate (panBC) synthesis. With this strain a D-pantothenate accumulation of up to 1 g/liter is achieved, which is a 10(5)-fold increase in concentration compared to that of the original wild-type strain. From the series of strains analyzed it follows that an increased ketoisovalerate availability is mandatory to direct the metabolite flux into the D-pantothenate-specific part of the pathway and that the availability of beta-alanine is essential for D-pantothenate formation.

Response of the central metabolism in Corynebacterium glutamicum to the use of an NADH-dependent glutamate dehydrogenase.

The extensive use of 13C enrichments in precursor metabolites for flux quantification does not rely on NADPH stoichiometries and can therefore be used to quantify reducing power fluxes. As an application of this concept, the NADPH fluxes were quantified in an L-lysine producer of Corynebacterium glutamicum grown into metabolic and isotopic steady state with [1-13C]glucose. In this case, where the organism's NADPH-dependent glutamate dehydrogenase consumes reducing power, the NADPH flux generated is 210% (molar flux relative to glucose uptake rate) with its major part (72% of the total) generated via the pentose phosphate pathway activity. An isogenic strain in which the glutamate dehydrogenase of C. glutamicum was replaced by the NADH-dependent glutamate dehydrogenase of Peptostreptococcus asaccharolyticus was made and the metabolite fluxes were again estimated. The major response to this local perturbation is a drastically reduced NADPH generation of only 139%. Most of the NADPH (62% of the total) is now generated via the tricarboxylic acid cycle activity. This shows the extraordinary flexibility of the central metabolism and provides a picture of the global regulatory properties of the central metabolism. Furthermore, a detailed analysis of the fluxes and exchange fluxes within the anaplerotic reactions is given. It is hypothesized that these reactions might also serve to balance the total reducing power budget as well as the energy budget within the cell.

The LysE superfamily: topology of the lysine exporter LysE of Corynebacterium glutamicum, a paradyme for a novel superfamily of transmembrane solute translocators.

In Corynebacterium glutamicum the LysE carrier protein exhibits the unique function of exporting L-lysine. We here analyze the membrane topology of LysE, a protein of 236 amino acyl residues, using PhoA- and LacZ-fusions. The amino-terminal end of LysE is located in the cytoplasm whereas the carboxy-terminal end is found in the periplasm. Although 6 hydrophobic domains were identified based on hydropathy analyses, only five transmembrane spanning helices appear to be present. The additional hydrophobic segment may dip into the membrane or be surface localized. We show that LysE is a member of a family of proteins found, for example, in Escherichia coil, Bacillus subtilis, Mycobacterium tuberculosis and Helicobacter pylori. This family, which we have designated the LysE family, is distantly related to two additional protein families which we have designated the YahN and CadD families. These three families, the members of which exhibit similar sizes, hydropathy profiles, and sequence motifs comprise the LysE superfamily. Functionally characterized members of the LysE superfamily export L-lysine, cadmium and possibly quarternary amines. We suggest that LysE superfamily members will prove to catalyze export of a variety of biologically important solutes.

Analysis of the leuB gene from Corynebacterium glutamicum.

The leuB gene of Corynebacterium glutamicum was found to be present on a 2.2-kb BamHI-SacI chromosomal fragment which complemented the leuB mutation of Escherichia coli. The activity of 3-isopropylmalate dehydrogenase (EC 1.1.1.85), encoded by the leuB gene, was significantly increased in C. glutamicum cells harbouring a plasmid containing the 2.2-kb fragment. The nucleotide sequence of the C. glutamicum leuB coding region (an open reading frame, ORF, of 1020 bp encoding a polypeptide of 340 amino acids with M(r) of 36 144) was determined. The deduced amino acid sequence of the product of this ORF is highly homologous to those of 3-isopropylmalate dehydrogenases from three species of mycobacteria. The transcriptional start site of the leuB gene was localized 35 bp upstream of its translational start; a functional terminator was detected in the 3' flanking region. Northern hybridization analysis showed that the C. glutamicum leuB gene is transcribed as a single monocistronic RNA (approximately 1.2 kb in size). Activity of the leuB promoter was significantly reduced when leucine was present in the growth medium. This suggests the negative regulation of the leuB expression on the transcriptional level in C. glutamicum cells.