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.

[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.

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.

Cloning, organization and functional analysis of ilvA, ilvB and ilvC genes from Corynebacterium glutamicum.

Corynebacterium glutamicum is an industrially important bacterium for the manufacture of amino acids. We constructed genomic libraries of this Gram+ bacterium and screened for clones carrying isoleucine biosynthesis genes (ilv) by complementation of Escherichia coli mutants. Clones complementing ilvA, ilvB, and ilvC were isolated. As based on the functional analysis of the corresponding plasmids in C. glutamicum, the DNA fragments isolated encode threonine dehydratase, acetohydroxy acid synthase, and isomeroreductase, catalyzing three subsequent reactions in Ile synthesis. Subcloning and transposon mutagenesis revealed that ilvB and ilvC reside on a 7-kb chromosomal fragment and that these genes are transcribed in the same direction. A shuttle vector was constructed to allow exonuclease treatment and assay subsets of plasmids for gene expression in the original C. glutamicum background. These constructs and their enzyme activity determinations revealed that despite close linkage ilvC is expressed independently from ilvB. Using Southern blots, a 15-kb fragment of chromosomal DNA carrying the ilvBC cluster was characterized. This fragment does not contain ilvA, demonstrating the entirely different organization of the isoleucine biosynthesis genes in C. glutamicum from that in enterobacteria.

Construction of L-lysine-, L-threonine-, and L-isoleucine-overproducing strains of Corynebacterium glutamicum.

The gram-negative bacterium Corynebacterium glutamicum is used for the industrial production of amino acids, for example, of L-glutamate and L-lysine. By cloning and expressing the various genes of the L-lysine pathway in C. glutamicum, we would demonstrate that an increase of the flux of L-aspartate semialdehyde to L-lysine could be obtained in strains with increased dihydrodipicolinate synthase activity. Recently we detected that in C. glutamicum two pathways exist for synthesis of D,L-diaminopimelate and L-lysine. Mutants defective in one pathway are still able to synthesize enough L-lysine for growth, but the L-lysine secretion is reduced to 50 to 70%. Using NMR spectroscopy, we could calculate how much of the L-lysine secreted into the medium is synthesized via either one or the other pathway. Amplification of the feedback inhibition insensitive homoserine dehydrogenase and homoserine kinase in a high L-lysine-overproducing strain enabled channeling of the carbon flow from the intermediate aspartate semialdehyde towards homoserine, resulting in a high accumulation of L-threonine. For a further flux from L-threonine to L-isoleucine, the allosteric control of threonine dehydratase was eliminated.

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.

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.

Biology of L-lysine overproduction byCorynebacterium glutamicum.

The Gram positive bacteriumCorynebacterium glutamicum is used for the production of L-lysine. This review focuses on the progress achieved in the past five years for a deeper understanding of lysine overproduction. This period also coincides with decisive progress in the use of genetic engineering techniques for analysing and increasing the metabolite flux inC. glutamicum. It was thus demonstrated that thein vivo activity of the allosterically controlled aspartate kinase is important for flux control, but in addition also the amount of the dihydrodipicolinate synthase. An outstanding feature ofC. glutamicum is the split lysine biosynthesis pathway. NMR investigations have clearly shown that both pathways are simultaneously usedin vivo and that the flux ratio depends on nitrogen availability. The cellular synthesized lysine is eventually exported into the external medium through a specific carrier. Interestingly lysine producers have other export characteristics so that the carrier properties also seem to be important for increased metabolite flux.

Regulation of alcohol oxidase synthesis in Hansenula polymorpha: oversynthesis during growth on mixed substrates and induction by methanol.

The regulation of the synthesis of alcohol oxidase, catalase, formaldehyde dehydrogenase and formate dehydrogenase was investigated in the methanol-utilizing yeast Hansenula polymorpha. The organism was found to synthesize immunologically identical alcohol oxidases during growth on glycerol and methanol. Growth on glycerol, however, was not dependent on the alcohol oxidase, as was shown with a mutant without alcohol oxidase protein. Similarly it was shown with a catalase activity negative mutant that high catalase activity during growth on glycerol was not a prerequisite for the utilization of this substrate, though absolutely required for growth on methanol. Experiments were conducted with mixed substrates to study the influence of methanol on alcohol oxidase synthesis. In batch cultures, growth on ribose plus methanol resulted in an enhanced rate of alcohol oxidase synthesis as compared to ribose alone. In continuous cultures, (D = 0.1 h-1) addition of methanol to glycerol-, glucose-, or sorbose-limited cultures gave rise to increased alcohol oxidase activity of up to 20 U/mg, which is about by 2 times higher than the specific activity used for growth on methanol alone. The increase in specific activity of the dissimilatory enzymes on the mixed substrates is partly due to methanol per se, as was shown by a mutant unable to dissimilate or assimilate methanol.

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.

Direct Enzymatic Assay for Alcohol Oxidase, Alcohol Dehydrogenase, and Formaldehyde Dehydrogenase in Colonies of Hansenula polymorpha.

A procedure is described for the qualitative direct identification of alcohol oxidase, alcohol dehydrogenase, and formaldehyde dehydrogenase in yeast colonies. The method has been applied successfully to isolate mutants of Hansenula polymorpha with altered glucose repression of alcohol oxidase.

The formaldehyde dehydrogenase of Rhodococcus erythropolis, a trimeric enzyme requiring a cofactor and active with alcohols.

During growth on compounds containing methyl groups a formaldehyde dehydrogenase is induced in the gram-positive bacteria Rhodococcus erythropolis. This formaldehyde dehydrogenase has been purified to homogeneity using affinity chromatography and permeation chromatography. The isoelectric point of the enzyme was 4.7. The molar mass of the native enzyme was determined as 130 000 g/mol. Sodium dodecyl sulfate gel electrophoresis yielded a single subunit with a molar mass of 44000 g/mol. These results, together with cross-linking experiments which yielded monomer, dimer, and trimer bands, are consistent with a trimeric subunit structure of the formaldehyde dehydrogenase. A heat-stable cofactor of low molar mass was required for activity with formaldehyde as substrate. This cofactor was found to be oxidizable, but active only in its reduced form. Preparative electrofocusing revealed that the cofactor is a weak acid with a pK of about 6.5. The enzyme was active with the homologous series of the primary alcohols, ethanol up to octanol, without requiring the presence of the cofactor. A mutant without formaldehyde dehydrogenase activity was not impaired in its growth with ethanol as substrate. It is suggested that the alcohols mimic the true substrate of the formaldehyde dehydrogenase, which could be a hydroxymethyl derivative of the cofactor, resulting from the addition of formaldehyde.

Threonine dehydratases of Corynebacterium glutamicum with altered allosteric control: their generation and biochemical and structural analysis.

Threonine dehydratase is the key enzyme in L-isoleucine synthesis, since it is allosterically feedback-inhibited by L-isoleucine. With the aim of obtaining regulatorily altered mutants of the threonine dehydratase of Corynebacterium glutamicum, amino acids were specifically exchanged and a new biological system of mutant selection was developed, based on the intoxication of Escherichia coli by ketobutyrate, which is the dehydratase reaction product. A collection of 19 mutant enzymes was generated and genetically and biochemically characterized comprising a whole range of regulatorily and catalytically altered enzymes. Of particular interest is the mutant Val-323-Ala, which is characterized by the fact that the L-isoleucine inhibition is entirely abolished so that the enzyme is always present in a relaxed, high-activity state. Correspondingly, the Hill coefficient is 1.4, in contrast to the value of 3.4 characteristic of the wild-type enzyme. Another peculiar mutant generated is the double mutant His-278-Arg-Leu-351-Ser. Here, again, L-isoleucine no longer inhibits catalytic activity, but the effector still promotes major structural changes of the protein, as ascertained from the L-isoleucine-dependent loss of pyridoxal-5'-phosphate from this mutant enzyme. Further enzymes obtained are reduced in L-isoleucine inhibition to a varying degree. Detailed studies on the structure of the enzyme revealed a partially very high similarity of the secondary structure to the mechanistically identical beta-subunit of the tryptophan synthase. This provides further indications concerning the localization of the regulatory and catalytic domain of the threonine dehydratase.

Use of Feedback-Resistant Threonine Dehydratases of Corynebacterium glutamicum To Increase Carbon Flux towards l-Isoleucine.

The biosynthesis of l-isoleucine proceeds via a highly regulated reaction sequence connected with l-lysine and l-threonine synthesis. Using defined genetic Corynebacterium glutamicum strains characterized by different fluxes through the homoserine dehydrogenase reaction, we analyzed the influence of four different ilvA alleles (encoding threonine dehydratase) in vectors with two different copy numbers on the total flux towards l-isoleucine. For this purpose, 18 different strains were constructed and analyzed. The result was that unlike ilvA in vectors with low copy numbers, ilvA in high-copy-number vectors increased the final l-isoleucine yield by about 20%. An additional 40% increase in l-isoleucine yield was obtained by the use of ilvA alleles encoding feedback-resistant threonine dehydratases. The strain with the highest yield was characterized by three hom(Fbr) copies encoding feedback-resistant homoserine dehydrogenase and ilvA(Fbr) encoding feedback-resistant threonine dehydratase on a multicopy plasmid. It accumulated 96 mM l-isoleucine, without any l-threonine as a by-product. The highest specific productivity was 0.052 g of l-isoleucine per g of biomass per h. This comparative flux analysis of isogenic strains showed that high levels of l-isoleucine formation from glucose can be achieved by the appropriate balance of homoserine dehydrogenase and threonine dehydratase activities in a strain background with feedback-resistant aspartate kinase. However, still-unknown limitations are present within the entire reaction sequence.

Strains of Corynebacterium glutamicum with Different Lysine Productivities May Have Different Lysine Excretion Systems.

The lysine excretion systems of three different lysine-producing strains of Corynebacterium glutamicum were characterized in intact cells. Two strains (DG 52-5 and MH 20-22B) are lysine producers of different efficiency. They were bred by classical mutagenesis and have a feedback-resistant aspartate kinase. The third strain (KK 25) was constructed from the wild type by introducing the feedback-resistant aspartate kinase gene of strain MH 20-22B into its genome. The three strains were shown to possess different excretion systems. Export in strain KK 25 is much slower than in the two mutants. The differences between the two lysine-producing strains are more subtle. K(m) and V(max) are similar, but pH dependence and membrane potential dependence reveal differences in the intrinsic properties of the carrier system.

Isoleucine synthesis in Corynebacterium glutamicum: molecular analysis of the ilvB-ilvN-ilvC operon.

Acetohydroxy acid synthase (AHAS) and isomeroreductase (IR) catalyze subsequent reactions in the flux of metabolites towards isoleucine, valine, leucine, and pantothenate. A 4,705-bp DNA fragment from Corynebacterium glutamicum known to code for AHAS and IR was sequenced and analyzed by Northern (RNA blot) analysis. As in other bacteria, the AHAS of this gram-positive organism is encoded by two genes, ilvB and ilvN. Gene disruption verified that these genes encode the single AHAS activity in C. glutamicum. The start of ilvB was determined by amino-terminal sequencing of a fusion peptide. By Northern analysis of the ilvBNC cluster, three in vivo transcripts of 3.9, 2.3, and 1.1 kb were identified, corresponding to ilvBNC, ilvNC, and ilvC messages, respectively. The ilvC transcript (encoding IR) was by far the most abundant one. With a clone from which the ilvB upstream regions had been deleted, only the ilvNC and ilvC transcripts were synthesized, and with a clone from which the ilvN upstream regions had been deleted, only the smallest ilvC transcript was formed. It is therefore concluded that in the ilv operon of C. glutamicum, three promoters are active. The amounts of the ilvBNC and ilvNC transcripts increased in response to the addition of alpha-ketobutyrate to the growth medium. This was correlated to an increase in specific AHAS activity, whereas IR activity was not increased because of the relatively large amount of the ilvC transcript present under all conditions assayed. Therefore, the steady-state level of the ilvBNC and ilvNC messages contributes significantly to the total activity of the single AHAS. The ilvC transcript of this operon, however, is regulated independently and present in a large excess, which is in accord with the constant IR activities determined.

Functional and structural analyses of threonine dehydratase from Corynebacterium glutamicum.

Threonine dehydratase activity is an important element in the flux control of isoleucine biosynthesis. The enzyme of Corynebacterium glutamicum demonstrates a marked sigmoidal dependence of initial velocity on the threonine concentration, a dependence that is consistent with substrate-promoted conversion of the enzyme from a low-activity to a high-activity conformation. In the presence of the negative allosteric effector isoleucine, the K0.5 increased from 21 to 78 mM and the cooperativity, as expressed by the Hill coefficient increased from 2.4 to 3.7. Valine promoted opposite effects: the K0.5 was reduced to 12 mM, and the enzyme exhibited almost no cooperativity. Sequence determination of the C. glutamicum gene for this enzyme revealed an open reading frame coding for a polypeptide of 436 amino acids. From this information and the molecular weight determination of the native enzyme, it follows that the dehydratase is a tetramer with a total mass of 186,396 daltons. Comparison of the deduced polypeptide sequence with the sequences of known threonine dehydratases revealed surprising differences from the C. glutamicum enzyme in the carboxy-terminal portion. This portion is greatly reduced in size, and a large gap of 95 amino acids must be introduced to achieve homology. Therefore, the C. glutamicum enzyme must be considered a small variant of threonine dehydratase that is typically controlled by isoleucine and valine but has an altered structure reflecting a topological difference in the portion of the protein most likely to be important for allosteric regulation.

l-Isoleucine Production with Corynebacterium glutamicum: Further Flux Increase and Limitation of Export.

The synthesis of l-isoleucine with Corynebacterium glutamicum involves 11 reaction steps, in at least five of which activity or expression is regulated. We used four genes and alleles encoding feedback-resistant enzymes (Fbr) in various combinations to assay flux increase through the sequence. During strain construction, the order of genes overexpressed was important. Only when ilvA(Fbr) was first overexpressed could hom(Fbr) be introduced. This succession apparently prevents the toxic accumulation of biosynthesis intermediates. The best strain constructed (SM13) was characterized by high-level expression of hom(Fbr), thrB, and ilvA(Fbr). With this strain a yield of 0.22 g of l-isoleucine per g of glucose was obtained, with a maximal specific productivity of 0.10 g of l-isoleucine per g (dry weight) per h. In strain SM13, with the high metabolite flux through the reaction sequence, effects on (i) other enzyme levels, (ii) time-dependent variations with process time, and (iii) concentrations of cytosolic intermediates were quantified. Most importantly, the intracellular l-isoleucine concentration is always higher at all process times than the extracellular concentration. The intracellular concentration rises to 110 mM, whereas extracellularly only 60 mM is accumulated. Also the immediate l-isoleucine precursor 2-ketomethyl valerate accumulates in the cell. Therefore, in the high-level l-isoleucine producer SM13, the export of this amino acid is the major limiting reaction step and therefore is a new target of strain design for biotechnological purposes.