Bicistronic expression of nuclear transgenes in Chlamydomonas reinhardtii.

In eukaryotic organisms, proteins are typically translated from monocistronic messenger RNAs containing a single coding sequence (CDS). However, recent long transcript sequencing identified 87 nuclear polycistronic mRNAs in Chlamydomonas reinhardtii natively carrying multiple co-expressed CDSs. In this study, we investigated the dynamics of 22 short intergenic sequences derived from these native polycistronic loci by their application in genetic constructs for synthetic transgene expression. A promising candidate sequence was identified based on the quantification of transformation efficiency and expression strength of a fluorescence reporter protein. Subsequently, the expression of independent proteins from one mRNA was verified by cDNA amplification and protein molecular mass characterization. We demonstrated engineered bicistronic expression in vivo to drive successful co-expression of several terpene synthases with the selection marker aphVIII. Bicistronic transgene design resulted in significantly increased (E)-α-bisabolene production of 7.95 mg L-1 from a single open reading frame, 18.1× fold higher than previous reports. Use of this strategy simplifies screening procedures for identification of high-level expressing transformants, does not require the application of additional fluorescence reporters, and reduces the nucleotide footprint compared to classical monocistronic expression cassettes. Although clear advantages for bicistronic transgene expression were observed, this strategy was found to be limited to the aphVIII marker, and further studies are necessary to gain insights into the underlying mechanism that uniquely permits this co-expression from the algal nuclear genome.

A gene regulatory network for antenna size control in carbon dioxide-deprived Chlamydomonas reinhardtii cells.

In green microalgae, prolonged exposure to inorganic carbon depletion requires long-term acclimation responses, involving modulated gene expression and the adjustment of photosynthetic activity to the prevailing supply of carbon dioxide. Here, we describe a microalgal regulatory cycle that adjusts the light-harvesting capacity at photosystem II (PSII) to the prevailing supply of carbon dioxide in Chlamydomonas (Chlamydomonas reinhardtii). It engages low carbon dioxide response factor (LCRF), a member of the squamosa promoter-binding protein (SBP) family of transcription factors, and the previously characterized cytosolic translation repressor nucleic acid-binding protein 1 (NAB1). LCRF combines a DNA-binding SBP domain with a conserved domain for protein-protein interaction. LCRF transcription is rapidly induced by carbon dioxide depletion. LCRF activates NAB1 transcription by specifically binding to tetranucleotide motifs present in its promoter. Accumulation of the NAB1 protein enhances translational repression of its prime target mRNA, encoding the PSII-associated major light-harvesting protein LHCBM6. The resulting truncation of the PSII antenna size helps maintaining a low excitation during carbon dioxide limitation. Analyses of low carbon dioxide acclimation in nuclear insertion mutants devoid of a functional LCRF gene confirm the essentiality of this novel transcription factor for the regulatory circuit.

Introns mediate post-transcriptional enhancement of nuclear gene expression in the green microalga Chlamydomonas reinhardtii.

Efficient nuclear transgene expression in the green microalga Chlamydomonas reinhardtii is generally hindered by low transcription rates. Introns can increase transcript abundance by a process called Intron-Mediated Enhancement (IME) in this alga and has been broadly observed in other eukaryotes. However, the mechanisms of IME in microalgae are poorly understood. Here, we identified 33 native introns from highly expressed genes in C. reinhardtii selected from transcriptome studies as well as 13 non-native introns. We investigated their IME capacities and probed the mechanism of action by modification of splice sites, internal sequence motifs, and position within transgenes. Several introns were found to elicit strong IME and found to be broadly applicable in different expression constructs. We determined that IME in C. reinhardtii exclusively occurs from introns within transcribed ORFs regardless of the promoter and is not induced by traditional enhancers of transcription. Our results elucidate some mechanistic details of IME in C. reinhardtii, which are similar to those observed in higher plants yet underly distinctly different induction processes. Our findings narrow the focus of targets responsible for algal IME and provides evidence that introns are underestimated regulators of C. reinhardtii nuclear gene expression.

Advanced pathway engineering for phototrophic putrescine production.

The polyamine putrescine (1,4-diaminobutane) contributes to cellular fitness in most organisms, where it is derived from the amino acids ornithine or arginine. In the chemical industry, putrescine serves as a versatile building block for polyamide synthesis. The green microalga Chlamydomonas reinhardtii accumulates relatively high putrescine amounts, which, together with recent advances in genetic engineering, enables the generation of a powerful green cell factory to promote sustainable biotechnology for base chemical production. Here, we report a systematic investigation of the native putrescine metabolism in C. reinhardtii, leading to the first CO2 -based bio-production of putrescine, by employing modern synthetic biology and metabolic engineering strategies. A CRISPR/Cas9-based knockout of key enzymes of the polyamine biosynthesis pathway identified ornithine decarboxylase 1 (ODC1) as a gatekeeper for putrescine accumulation and demonstrated that the arginine decarboxylase (ADC) route is likely inactive and that amine oxidase 2 (AMX2) is mainly responsible for putrescine degradation in C. reinhardtii. A 4.5-fold increase in cellular putrescine levels was achieved by engineered overexpression of potent candidate ornithine decarboxylases (ODCs). We identified unexpected substrate promiscuity in two bacterial ODCs, which exhibited co-production of cadaverine and 4-aminobutanol. Final pathway engineering included overexpression of recombinant arginases for improved substrate availability as well as functional knockout of putrescine degradation, which resulted in a 10-fold increase in cellular putrescine titres and yielded 200 mg/L in phototrophic high cell density cultivations after 10 days.

Engineering a powerful green cell factory for robust photoautotrophic diterpenoid production.

Diterpenoids display a large and structurally diverse class of natural compounds mainly found as specialized plant metabolites. Due to their diverse biological functions they represent an essential source for various industrially relevant applications as biopharmaceuticals, nutraceuticals, and fragrances. However, commercial production utilizing their native hosts is inhibited by low abundances, limited cultivability, and challenging extraction, while the precise stereochemistry displays a particular challenge for chemical synthesis. Due to a high carbon flux through their native 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway towards photosynthetically active pigments, green microalgae hold great potential as efficient and sustainable heterologous chassis for sustainable biosynthesis of plant-derived diterpenoids. In this study, innovative synthetic biology and efficient metabolic engineering strategies were systematically combined to re-direct the metabolic flux through the MEP pathway for efficient heterologous diterpenoid synthesis in C. reinhardtii. Engineering of the 1-Deoxy-D-xylulose 5-phosphate synthase (DXS) as the main rate-limiting enzyme of the MEP pathway and overexpression of diterpene synthase fusion proteins increased the production of high-value diterpenoids. Applying fully photoautotrophic high cell density cultivations demonstrate potent and sustainable production of the high-value diterpenoid sclareol up to 656 mg L-1 with a maximal productivity of 78 mg L-1 day-1 in a 2.5 L scale photobioreactor, which is comparable to sclareol titers reached by highly engineered yeast. Consequently, this work represents a breakthrough in establishing a powerful phototrophic green cell factory for the competetive use in industrial biotechnology.

Turning a green alga red: engineering astaxanthin biosynthesis by intragenic pseudogene revival in Chlamydomonas reinhardtii.

The green alga Chlamydomonas reinhardtii does not synthesize high-value ketocarotenoids like canthaxanthin and astaxanthin; however, a ß-carotene ketolase (CrBKT) can be found in its genome. CrBKT is poorly expressed, contains a long C-terminal extension not found in homologues and likely represents a pseudogene in this alga. Here, we used synthetic redesign of this gene to enable its constitutive overexpression from the nuclear genome of C. reinhardtii. Overexpression of the optimized CrBKT extended native carotenoid biosynthesis to generate ketocarotenoids in the algal host causing noticeable changes the green algal colour to reddish-brown. We found that up to 50% of native carotenoids could be converted into astaxanthin and more than 70% into other ketocarotenoids by robust CrBKT overexpression. Modification of the carotenoid metabolism did not impair growth or biomass productivity of C. reinhardtii, even at high light intensities. Under different growth conditions, the best performing CrBKT overexpression strain was found to reach ketocarotenoid productivities up to 4.3 mg/L/day. Astaxanthin productivity in engineered C. reinhardtii shown here might be competitive with that reported for Haematococcus lacustris (formerly pluvialis) which is currently the main organism cultivated for industrial astaxanthin production. In addition, the extractability and bio-accessibility of these pigments were much higher in cell wall-deficient C. reinhardtii than the resting cysts of H. lacustris. Engineered C. reinhardtii strains could thus be a promising alternative to natural astaxanthin producing algal strains and may open the possibility of other tailor-made pigments from this host.

Intron-containing algal transgenes mediate efficient recombinant gene expression in the green microalga Chlamydomonas reinhardtii.

Among green freshwater microalgae, Chlamydomonas reinhardtii has the most comprehensive and developed molecular toolkit, however, advanced genetic and metabolic engineering driven from the nuclear genome is generally hindered by inherently low transgene expression levels. Progressive strain development and synthetic promoters have improved the capacity of transgene expression; however, the responsible regulatory mechanisms are still not fully understood. Here, we elucidate the sequence specific dynamics of native regulatory element insertion into nuclear transgenes. Systematic insertions of the first intron of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit 2 (rbcS2i1) throughout codon-optimized coding sequences (CDS) generates optimized algal transgenes which express reliably in C. reinhardtii. The optimal rbcS2i1 insertion site for efficient splicing was systematically determined and improved gene expression rates were shown using a codon-optimized sesquiterpene synthase CDS. Sequential insertions of rbcS2i1 were found to have a step-wise additive effect on all levels of transgene expression, which is likely correlated to a synergy of transcriptional machinery recruitment and mimicking the short average exon lengths natively found in the C. reinhardtii genome. We further demonstrate the value of this optimization with five representative transgene examples and provide guidelines for the design of any desired sequence with this strategy.

Tailored carbon partitioning for phototrophic production of (E)-α-bisabolene from the green microalga Chlamydomonas reinhardtii.

Photosynthetic microbial hosts such as cyanobacteria and eukaryotic microalgae have recently emerged as alternative engineering platforms for the sustainable light-driven bio-production of terpenoids. Many desirable compounds with numerous applications can be produced in microorganisms by heterologous expression of terpene synthases. However, success of green microbial systems has been hampered by issues such as insufficient enzyme expression titers and low flux to desired terpenoid products from carbon fixed during photosynthesis. This work demonstrates how the green microalga Chlamydomonas reinhardtii can be engineered to produce the sesquiterpene biodiesel precursor (E)-α-bisabolene. Through strategic genetic engineering, substantial enhancements of productivity were achieved by coordinated tuning of the isoprenoid metabolism, combining serial enzyme loading for terpene synthase overexpression and amiRNA-based repression of competing pathways. Up to 10.3 ± 0.7mg bisabolene·g-1 cell dry weight could be produced in five days, which represents more than a 15-fold increase over single synthase expression strains. Investigation of strain performance in scale-up cultivations determined overall bisabolene productivity benefits from light:dark cycles. Mixotrophic cultivation can yield up to 11.0 ± 0.5mg bisabolene per liter in seven days in these conditions, and phototrophic production of 3.9 ± 0.2mg per liter was feasible. These achievements represent an important milestone in the engineering of C. reinhardtii towards the goal of designing sustainable, light-driven, green-cell algal bio-factories.

Phototrophic production of heterologous diterpenoids and a hydroxy-functionalized derivative from Chlamydomonas reinhardtii.

Photosynthetic microalgae harbor enormous potential as light-driven green-cell factories for sustainable bio-production of a range of natural and heterologous products such as isoprenoids. Their capacity for photosynthesis and rapid low-input growth with (sun)light and CO2 is coupled to a robust metabolic architecture structured toward the generation of isoprenoid pigments and compounds involved in light capture, electron transfer, and radical scavenging. Metabolic engineering approaches using eukaryotic green microalgae have previously been hampered mainly by low-levels of nuclear transgene expression. Here, we employed a strategy of optimized transgene design which couples codon optimization and synthetic intron spreading for the expression of heterologous plant enzymes from the algal nuclear genome. The diterpenoids casbene, taxadiene, and 13R(+) manoyl oxide were produced after expressing heterologous diterpene synthases and enzymes participating in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway which were all targeted to the algal chloroplast. Additionally, a truncated and soluble plant microsomal cytochrome P450 monooxygenase was functionally expressed and able to hydroxylate 13R(+) manoyl oxide when directed into the chloroplasts. The heterologous diterpenoids were found to be excreted from the cells and accumulate in dodecane solvent-culture overlays. It was shown that the algal cell could tolerate significant metabolic pull towards diterpenoids without loss of native pigments. Using an algal strain producing 13R(+) manoyl oxide as a model, diterpenoid production was shown to be highest in photoautotrophic cultivations using CO2 as the sole carbon source and day:night illumination cycles. Up to 80 mg 13R(+) manoyl oxide per gram cell dry mass (CDM) could be produced from C. reinhardtii in a 7 day batch cultivation with a sustained maximal productivity of 22.5 mg gcdm-1 d-1 over 3 consecutive days. Collectively the results presented here suggest that green algal cells have remarkable potential for the heterologous production of non-native isoprenoids and support the use of these hosts for (sun)light driven bioproduction concepts.

Matching events and activities by integrating behavioral aspects and label analysis.

Nowadays, business processes are increasingly supported by IT services that produce massive amounts of event data during the execution of a process. These event data can be used to analyze the process using process mining techniques to discover the real process, measure conformance to a given process model, or to enhance existing models with performance information. Mapping the produced events to activities of a given process model is essential for conformance checking, annotation and understanding of process mining results. In order to accomplish this mapping with low manual effort, we developed a semi-automatic approach that maps events to activities using insights from behavioral analysis and label analysis. The approach extracts Declare constraints from both the log and the model to build matching constraints to efficiently reduce the number of possible mappings. These mappings are further reduced using techniques from natural language processing, which allow for a matching based on labels and external knowledge sources. The evaluation with synthetic and real-life data demonstrates the effectiveness of the approach and its robustness toward non-conforming execution logs.

Efficient phototrophic production of a high-value sesquiterpenoid from the eukaryotic microalga Chlamydomonas reinhardtii.

The heterologous expression of terpene synthases in microbial hosts has opened numerous possibilities for bioproduction of desirable metabolites. Photosynthetic microbial hosts present a sustainable alternative to traditional fermentative systems, using freely available (sun)light and carbon dioxide as inputs for bio-production. Here, we report the expression of a patchoulol synthase from Pogostemon cablin Benth in the model green microalga Chlamydomonas reinhardtii. The sesquiterpenoid patchoulol was produced from the alga and was used as a marker of sesquiterpenoid production capacity. A novel strategy for gene loading was employed and patchoulol was produced up to 922±242µgg-1 CDW in six days. We additionally investigated the effect of carbon source on sesquiterpenoid productivity from C. reinhardtii in scale-up batch cultivations. It was determined that up to 1.03mgL-1 sesquiterpenoid products could be produced in completely photoautotrophic conditions and that the alga exhibited altered sesquiterpenoid production metabolism related to carbon source.

CRISPR/Cas9-Mediated Knockout of the Lycopene ε-Cyclase for Efficient Astaxanthin Production in the Green Microalga Chlamydomonas reinhardtii.

Carotenoids are valuable pigments naturally occurring in all photosynthetic plants and microalgae as well as in selected fungi, bacteria, and archaea. Green microalgae developed a complex carotenoid profile suitable for efficient light harvesting and light protection and harbor great capacity for carotenoid production through the substantial power of the endogenous 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Previous works established successful genome editing and induced significant changes in the cellular carotenoid content in Chlamydomonas reinhardtii. This study employs a tailored carotenoid pathway for engineered bioproduction of the valuable ketocarotenoid astaxanthin. Functional knockout of lycopene ε-cyclase (LCYE) and non-homologous end joining (NHEJ)-based integration of donor DNA at the target site inhibit the accumulation of α-carotene and consequently lutein and loroxanthin, abundant carotenoids in C. reinhardtii without changes in cellular fitness. PCR-based screening indicated that 4 of 96 regenerated candidate lines carried (partial) integrations of donor DNA and increased ß-carotene as well as derived carotenoid contents. Iterative overexpression of CrBKT, PacrtB, and CrCHYB resulted in a 2.3-fold increase in astaxanthin accumulation in mutant ΔLCYE#3 (1.8 mg/L) compared to the parental strain UVM4, which demonstrates the potential of genome editing for the design of a green cell factory for astaxanthin bioproduction.

Outdoor cultivation and metabolomics exploration of Chlamydomonas engineered for bisabolene production.

Demonstrating outdoor cultivation of engineered microalgae at considerable scales is essential for their prospective large-scale deployment. Hence, this study focuses on the outdoor cultivation of an engineered Chlamydomonas reinhardtii strain, 3XAgBs-SQs, for bisabolene production under natural dynamic conditions of light and temperature. Our preliminary outdoor experiments showed improved growth, but frequent culture collapses in conventional Tris-acetate-phosphate medium. In contrast, modified high-salt medium (HSM) supported prolonged cell survival, outdoor. However, their subsequent outdoor scale-up from 250 mL to 5 L in HSM was effective with 10 g/L bicarbonate supplementation. Pulse amplitude modulation fluorometry and metabolomic analysis further validated their improved photosynthesis and uncompromised metabolic fluxes towards the biomass and the products (natural carotenoids and engineered bisabolene). These strains could produce 906 mg/L bisabolene and 54 mg/L carotenoids, demonstrating the first successful outdoor photoautotrophic cultivation of engineeredC. reinhardtii,establishing it as a one-cell two-wells biorefinery.

Modular slot-die coater for in situ grazing-incidence x-ray scattering experiments on thin films.

Multimodal in situ experiments during slot-die coating of thin films pioneer the way to kinetic studies on thin-film formation. They establish a powerful tool to understand and optimize the formation and properties of thin-film devices, e.g., solar cells, sensors, or LED films. Thin-film research benefits from time-resolved grazing-incidence wide- and small-angle x-ray scattering (GIWAXS/GISAXS) with a sub-second resolution to reveal the evolution of crystal structure, texture, and morphology during the deposition process. Simultaneously investigating optical properties by in situ photoluminescence measurements complements in-depth kinetic studies focusing on a comprehensive understanding of the triangular interdependency of processing, structure, and function for a roll-to-roll compatible, scalable thin-film deposition process. Here, we introduce a modular slot-die coater specially designed for in situ GIWAXS/GISAXS measurements and applicable to various ink systems. With a design for quick assembly, the slot-die coater permits the reproducible and comparable fabrication of thin films in the lab and at the synchrotron using the very same hardware components, as demonstrated in this work by experiments performed at Deutsches Elektronen-Synchrotron (DESY). Simultaneous to GIWAXS/GISAXS, photoluminescence measurements probe optoelectronic properties in situ during thin-film formation. An environmental chamber allows to control the atmosphere inside the coater. Modular construction and lightweight design make the coater mobile, easy to transport, quickly extendable, and adaptable to new beamline environments.

Current Nuclear Engineering Strategies in the Green Microalga Chlamydomonas reinhardtii.

The green model microalga Chlamydomonas reinhardtii recently emerged as a sustainable production chassis for the efficient biosynthesis of recombinant proteins and high-value metabolites. Its capacity for scalable, rapid and light-driven growth in minimal salt solutions, its simplicity for genetic manipulation and its "Generally Recognized As Safe" (GRAS) status are key features for its application in industrial biotechnology. Although nuclear transformation has typically resulted in limited transgene expression levels, recent developments now allow the design of powerful and innovative bioproduction concepts. In this review, we summarize the main obstacles to genetic engineering in C. reinhardtii and describe all essential aspects in sequence adaption and vector design to enable sufficient transgene expression from the nuclear genome. Several biotechnological examples of successful engineering serve as blueprints for the future establishment of C. reinhardtii as a green cell factory.

Molecular design of microalgae as sustainable cell factories.

Microalgae are regarded as sustainable and potent chassis for biotechnology. Their capacity for efficient photosynthesis fuels dynamic growth independent from organic carbon sources and converts atmospheric CO2 directly into various valuable hydrocarbon-based metabolites. However, approaches to gene expression and metabolic regulation have been inferior to those in more established heterotrophs (e.g., prokaryotes or yeast) since the genetic tools and insights in expression regulation have been distinctly less advanced. In recent years, however, these tools and their efficiency have dramatically improved. Various examples have demonstrated new trends in microalgal biotechnology and the potential of microalgae for the transition towards a sustainable bioeconomy.

Metabolic Engineering for Efficient Ketocarotenoid Accumulation in the Green Microalga Chlamydomonas reinhardtii.

Astaxanthin is a valuable ketocarotenoid with various pharmaceutical and nutraceutical applications. Green microalgae harbor natural capacities for pigment accumulation due to their 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Recently, a redesigned ß-carotene ketolase (BKT) was found to enable ketocarotenoid accumulation in the model microalga Chlamydomonas reinhardtii, and transformants exhibited reduced photoinhibition under high-light. Here, a systematic screening by synthetic transgene design of carotenoid pathway enzymes and overexpression from the nuclear genome identified phytoene synthase (PSY/crtB) as a bottleneck for carotenoid accumulation in C. reinhardtii. Increased ß-carotene hydroxylase (CHYB) activity was found to be essential for engineered astaxanthin accumulation. A combined BKT, crtB, and CHYB expression strategy resulted in a volumetric astaxanthin production of 9.5 ± 0.3 mg L-1 (4.5 ± 0.1 mg g-1 CDW) in mixotrophic and 23.5 mg L-1 (1.09 mg L-1 h-1) in high cell density conditions, a 4-fold increase compared to previous reports in C. reinhardtii. This work presents a systematic investigation of bottlenecks in astaxanthin accumulation in C. reinhardtii and the phototrophic green cell factory design for competitive use in industrial biotechnology.

Engineering astaxanthin accumulation reduces photoinhibition and increases biomass productivity under high light in Chlamydomonas reinhardtii.

BACKGROUND: Astaxanthin is a highly valuable ketocarotenoid with strong antioxidative activity and is natively accumulated upon environmental stress exposure in selected microorganisms. Green microalgae are photosynthetic, unicellular organisms cultivated in artificial systems to produce biomass and industrially relevant bioproducts. While light is required for photosynthesis, fueling carbon fixation processes, application of high irradiance causes photoinhibition and limits biomass productivity. RESULTS: Here, we demonstrate that engineered astaxanthin accumulation in the green alga Chlamydomonas reinhardtii conferred high light tolerance, reduced photoinhibition and improved biomass productivity at high irradiances, likely due to strong antioxidant properties of constitutively accumulating astaxanthin. In competitive co-cultivation experiments, astaxanthin-rich Chlamydomonas reinhardtii outcompeted its corresponding parental background strain and even the fast-growing green alga Chlorella vulgaris. CONCLUSIONS: Metabolic engineering inducing astaxanthin and ketocarotenoids accumulation caused improved high light tolerance and increased biomass productivity in the model species for microalgae Chlamydomonas reinhardtii. Thus, engineering microalgal pigment composition represents a powerful strategy to improve biomass productivities in customized photobioreactors setups. Moreover, engineered astaxanthin accumulation in selected strains could be proposed as a novel strategy to outperform growth of other competing microalgal strains.

The Spermidine Synthase Gene SPD1: A Novel Auxotrophic Marker for Chlamydomonas reinhardtii Designed by Enhanced CRISPR/Cas9 Gene Editing.

Biotechnological application of the green microalga Chlamydomonas reinhardtii hinges on the availability of selectable markers for effective expression of multiple transgenes. However, biological safety concerns limit the establishment of new antibiotic resistance genes and until today, only a few auxotrophic markers exist for C. reinhardtii. The recent improvements in gene editing via CRISPR/Cas allow directed exploration of new endogenous selectable markers. Since editing frequencies remain comparably low, a Cas9-sgRNA ribonucleoprotein (RNP) delivery protocol was strategically optimized by applying nitrogen starvation to the pre-culture, which improved successful gene edits from 10% to 66% after pre-selection. Probing the essential polyamine biosynthesis pathway, the spermidine synthase gene (SPD1) is shown to be a potent selectable marker with versatile biotechnological applicability. Very low levels of spermidine (0.75 mg/L) were required to maintain normal mixotrophic and phototrophic growth in newly designed spermidine auxotrophic strains. Complementation of these strains with a synthetic SPD1 gene was achieved when the mature protein was expressed in the cytosol or targeted to the chloroplast. This work highlights the potential of new selectable markers for biotechnology as well as basic research and proposes an effective pipeline for the identification of new auxotrophies in C. reinhardtii.

Flap irritation phenomenon (FLIP): etiology of chronic tenosynovitis after finger pulley rupture.

After a pulley rupture, most climbers regain the full function of their previously uninjured fingers. However, in some cases of pulley rupture, a persistent inflammation of the tendon sheath is observed. In this study, 16 cadaver fingers were loaded until pulley rupture and then studied for the rupturing mechanism. In addition, two patients with this pathology were investigated using ultrasound and MRI, and received surgery. In 13 fingers, a rupture of one or several pulleys occurred and almost always at the medial or lateral insertion. In one finger, a capsizing of the pulley underneath the intact tendon sheath was observed, leading to an avulsion between tendon and tendon sheath. A similar pathology was observed in the ultrasound imaging, in MRI, and during surgery in two patients with prolonged recovery after minor pulley rupture. In cases of prolonged tenosynovitis after minor pulley rupture, a capsizing of the pulley stump is probably the cause for constant friction leading to inflammation. In those cases, a surgical removal of the remaining pulley stump and sometimes a pulley repair may be necessary.