Propionibacterium acnes endocarditis: a case series.

OBJECTIVES: Propionibacterium acnes remains a rare cause of infective endocarditis (IE). It is challenging to diagnose due to the organism's fastidious nature and the indolent presentation of the disease. The purpose of this study was to describe the clinical presentation and management of P. acnes IE with an emphasis on the methods of diagnosis. METHODS: We identified patients from the Cleveland Clinic Infective Endocarditis Registry who were admitted from 2007 to 2015 with definite IE by Duke Criteria. Propionibacterium acnes was defined as the causative pathogen if it was identified in at least two culture specimens, or identified with at least two different modalities: blood culture, valve culture, valve sequencing or histopathological demonstration of microorganisms. RESULTS: We identified 24 cases of P. acnes IE, 23 (96%) of which were either prosthetic valve endocarditis or IE on an annuloplasty ring. Invasive disease (71%) and embolic complications (29%) were common. All but one patient underwent surgery. Propionibacterium acnes was identified in 12.5% of routine blood cultures, 75% of blood cultures with extended incubation, 55% of valve cultures, and 95% of valve sequencing specimens. In 11 of 24 patients (46%), no causative pathogen would have been identified without valve sequencing. CONCLUSIONS: Propionibacterium acnes almost exclusively causes prosthetic valve endocarditis and patients often present with advanced disease. The organism may not be readily cultured, and extended cultures appear to be necessary. In patients who have undergone surgery, valve sequencing is most reliable in establishing the diagnosis.

Genetic evaluation of in-line recorded milkability from milking parlors and automatic milking systems.

The overall objective of this study was to assess the use of in-line recorded milkability information from dairy herds with conventional milking parlors (CMP) and from herds with automatic milking systems (AMS) for genetic evaluation. Some genetic parameters were previously studied on AMS data for 2,053 Swedish Holstein (SH) and 1,749 Swedish Red (SR) cows in 19 herds. These data were combined in the present paper with milkability information from 74 herds with CMP, including 11,123 SH cows and 7,554 SR cows. Genetic parameters were estimated for the CMP data and genetic correlations were estimated between milkability traits measured in the 2 systems. Average flow rate and milking time were derived and used as similar milkability traits for both systems, whereas box time was used only for AMS herds. Estimated heritabilities were in the range from 0.24 to 0.49. Even though the traits were differently defined in the 2 milking systems, the corresponding traits recorded in AMS and CMP were genetically closely related (0.93-1.00). Similarly, close genetic relationships were shown between milkability traits in different lactations in both breeds (0.93-0.99). Thus, it should be possible to treat milkability traits in different lactations and from different milking systems as the same traits in genetic evaluations. The various milkability traits were also highly genetically correlated, indicating that the inclusion of just one trait in the genetic selection program would efficiently select for milkability without the need to consider all measures. Comparisons of repeatability and random regression models, combining all information from the 2 systems for genetic evaluation, were done to find the most suitable model for genetic evaluation purposes. Even though the random regression models were favored in the formal model tests to evaluate suitability, correlation coefficients between test-days within lactation were high (0.7-0.8) and small differences in breeding values resulted among different models. That would indicate that a few test-days per cow would produce accurate breeding values for milkability.

Feasibility of using automatic milking system data from commercial herds for genetic analysis of milkability.

The objective of this study was to investigate how useful data from automatic milking systems used in commercial herds are for genetic analysis of milkability traits. Data were available from 4,968 Swedish Holstein and Swedish Red cows over a span of 5 yr (2004-2009) from 19 herds. The analyzed milkability traits were average flow rate, box time, milking interval, and number of milkings per day. Variance components were estimated for genetic, permanent environmental, and residual effects in first and later (second and third) lactations, and were used for estimation of heritabilities and repeatablilites. The experiences of the data quality and editing procedures showed that almost half of the data and about a quarter of the cows had to be excluded from the analyses due to incomplete or inconsistent information. However, much more data are available than is needed for accurate genetic parameter estimations. For the genetic analysis, a repeatability animal model was used that included the fixed effects of herd, year and season, lactation month, and milk yield. The repeatability coefficients were at a high level: highest for average flow rate, with estimates between 0.8 and 0.9. The estimated heritability coefficients were in the range of 0.37 to 0.48, 0.21 to 0.44, 0.09 to 0.26, and 0.02 to 0.07 for average flow rate, box time, milking interval, and number of milkings, respectively. The results from the present study unraveled large genetic variation in milkability traits. The genetic parameter estimates were well in agreement with previous studies of milkability, which proves the feasibility of using data from automatic milking systems for genetic analysis.

Lung transplantation in a recipient with novel 2009 H1N1 influenza: lessons learned.

Immunosuppressed patients are at an increased risk for severe disease from influenza A (H1N1). We report a case of a patient who died of septic complications from H1N1 acquired at the time of single lung transplant.

Mid-luteal progesterone concentrations are associated with live birth rates during ovulation induction.

This retrospective study investigated whether mid-luteal serum progesterone concentrations are associated with live birth rates in women with WHO group II anovulatory infertility undergoing ovulation induction. Data were from women (n=335) stimulated with gonadotrophins using a low-dose step-up protocol, of which women with presumptive ovulation (n=279), defined as a mid-luteal progesterone concentration ⩾7.9ng/ml (⩾25nmol/l; range 7.9-194ng/ml) were included. Of the women with presumptive ovulation, 57 (20.4%) had a live birth and their serum mid-luteal progesterone concentration was significantly (P=0.016) higher than that of the non-live birth group. There were significant associations between the number of large (⩾15mm) and medium-sized follicles (12-14mm) at human chorionic gonadotrophin administration and the mid-luteal progesterone concentration (P<0.001), while the total number of large and medium-sized follicles was not significantly associated with live birth rate. In conclusion, mid-luteal progesterone concentrations above the cut-off values currently used for defining ovulation were positively associated with live birth rates in normogonadotrophic anovulatory women undergoing ovulation induction with gonadotrophins. The mid-luteal progesterone concentration, apart from being a consequence of the number of corpora lutea, may also reflect the quality of the follicle/oocyte/corpus luteum. Measurement of blood concentration of the steroid hormone progesterone in the mid-postovulatory phase of the menstrual cycle is frequently used to determine ovulation. The aim of this study was to investigate whether increasing blood concentrations of progesterone in the mid-postovulatory phase was associated with higher chances of achieving a live birth in a group of 335 women with anovulatory infertility, who had undergone stimulation with gonadotrophin hormones for the purpose of inducing ovulation. Statistical analysis, performed on the 279 women with presumptive ovulation (defined as a mid-postovulatory progesterone concentration ⩾7.9ng/ml serum), showed that the mid-postovulatory progesterone concentration was significantly positively associated with live birth rate. There was also a significant association between follicular development at end of gonadotrophin stimulation and the mid-postovulatory progesterone concentration, but follicular development could not explain live birth rate as mid-postovulatory progesterone concentrations could. In conclusion, increased blood concentrations of progesterone in the mid-postovulatory phase of the menstrual cycle above the threshold values currently used for defining ovulation were associated with increased live birth rates in anovulatory women undergoing ovulation induction with gonadotrophin hormones. The mid-postovulatory progesterone concentration, apart from being a consequence of the quantity of follicular development, may therefore also reflect the quality of the ovarian follicles and eggs.

Allograft entrapment after lung transplantation: a simple solution using a pleurocutaneous catheter.

BACKGROUND: Chronic pleural effusion following lung transplantation (LTx) is often responsible for respiratory insufficiency and can lead to lung entrapment. Decortication carries considerable morbidity, and extended use of tube thoracostomy is not practical. We have utilized an indwelling pleurocutaneous catheter in the setting of intractable post-transplant effusion and have reviewed our experience to determine whether this strategy: 1) facilitates resolution of effusion, and 2) adequately palliates lung entrapment. METHODS: Twelve PleurX (Denver Biomedical, Golden, CO, USA) catheters were placed in 9 LTx patients (6 unilateral, 3 bilateral) for refractory pleural effusions after standard tube thoracostomy drainage failed (12/12). Two-thirds of the patients (8/12) also had concomitant lung entrapment. RESULTS: There was no operative mortality. Median time from LTx to catheter placement was 79 days (range 21-769). Catheter use achieved the desired outcome in 11/12 placements. Catheters remained in place for a median of 86 days (range 35-190). Direct catheter-related complications included hemothorax (1) and empyema (1). CONCLUSION: Use of an indwelling pleurocutaneous catheter effectively achieves its intended goals of pleurodesis and management of entrapped lungs after LTx.

Prestimulation parameters predicting live birth in anovulatory WHO Group II patients undergoing ovulation induction with gonadotrophins.

BACKGROUND: The objective of this study was to identify baseline predictors of live birth in anovulatory patients undergoing ovulation induction, and based on these predictors, develop nomograms for estimation of the probability of live birth in a single cycle. METHODS: Univariate and multivariate logistic regression were used for retrospective analysis of clinical, sonographic and endocrinological parameters collected prior to the start of ovarian stimulation in a cohort of anovulatory World Health Organization (WHO) Group II patients (n = 335), who were resistant to clomiphene citrate (CC) and therefore stimulated with gonadotrophins using a low-dose step-up protocol. RESULTS: The univariate analysis identified age [OR = 0.91 (95% CI: 0.84-0.98), P = 0.015], duration of infertility [OR = 0.71 (95% CI: 0.56-0.91), P = 0.007], serum follicle stimulating hormone (FSH) concentration at the start of stimulation [OR = 0.83 (95% CI: 0.69-0.99), P = 0.034] and menstrual cycle pattern (P = 0.022) as significant predictors of live birth. Baseline concentrations of luteinizing hormone, androgens, glucose and insulin, as well as body mass index, were not predictors of live birth. In the multivariate analysis, duration of infertility, FSH and menstrual cycle pattern were independent predictors, and nomograms were designed with these three parameters for individual prediction of the probability of live birth. CONCLUSIONS: The chances of live birth in women with WHO Group II anovulatory infertility resistant to CC undergoing ovulation induction with gonadotrophins is highly influenced by the menstrual cycle pattern. Increases in duration of infertility and concentration of FSH (within the normal range) before the start of stimulation have negative influences on the likelihood of achieving a live birth.

Technical note: variation in daily milk yield calculations for dairy cows milked in an automatic milking system.

An accurate estimation of the daily milk yield of dairy cows milked in an automatic milking system is not obvious because of variations in milking intervals and frequencies. Daily harvested milk varies substantially, and developing a method to be used for estimating daily milk production is of great importance. Three calculation methods (simple, semiadvanced, and advanced) were used. The simple method calculated rough daily milk production by summing up the yield per day. The semiadvanced used yield in combination with time since last milking to calculate the milk production per hour between milking; an average of the milk production per hour over the day was calculated and multiplied by 24. The advanced method calculated the milk production from midnight to midnight by using information about yield and time since last milking to calculate the exact milk production. The results show a clear preference for the advanced calculation method because the variation [variation for the advanced method=ln(1.79) for first lactation and ln(2.28) for later lactations] between days was reduced significantly (3 to 4 times lower compared with the simple method). Variation in daily harvested milk can be used as a management tool.

Challenges in the diagnosis and management of Nocardia infections in lung transplant recipients.

BACKGROUND: Nocardia infection occurs in 2.1-3.5% of lung transplant recipients, and may involve cavitary nodular pulmonary lesions, soft tissue infection, or other sites of dissemination. Nocardiosis can pose challenging clinical problems in the areas of diagnosis and treatment. Diagnostic delays may occur, and adverse reactions to therapy are common. This study reviews clinical and epidemiological aspects of nocardiosis in lung transplant recipients, with special attention to pitfalls in management. Clinicians should be alert for these possibilities in order to institute prompt therapy and to achieve successful outcomes. METHODS: A retrospective cohort study was conducted of 577 lung transplant recipients from January 1991 to May 2007. Demographics, reason for transplant, recent rejection, time from transplantation, site of infection, hypogammaglobulinemia, and/or neutropenia shortly before onset, Pneumocystis jiroveci prophylaxis, Nocardia species, radiographic findings, extrapulmonary lesions, nature and duration of treatment, adverse reactions, and outcomes were recorded. RESULT: Nocardia infection occurred in 1.9% (11/577). Mean onset was 14.3 months after transplant (range 1.5-39 months). N. asteroides was isolated in 55% (6/11). Emphysema was the most common reason for transplant (7/11, 64%). Six patients were receiving trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis at the time of diagnosis. Three patients had immune globulin G levels <400 mg/dL and 2 were neutropenic in the 3 months preceding diagnosis. Diagnosis was made by bronchoalveolar lavage (55%), skin abscess culture (18%), open lung biopsy (9%), pleural fluid (9%), and sputum culture (9%). Definitive diagnosis required a median of 9 days and a mean of 13.6 days (range 3-35 days) from the time of diagnostic sampling. Soft tissue lesions occurred in 3 and central nervous system involvement in 1 patient. Adverse reactions to therapy occurred in 9/10 (90%) of patients for whom information was available. Nocardia-related mortality occurred in 2/11 patients (18%). CONCLUSIONS: Nocardiosis occurred in 1.9% of lung transplant recipients and was associated with a mean of nearly 2 weeks to diagnosis and frequent adverse effects on therapy. TMP-SMX prophylaxis on a thrice weekly basis did not prevent all episodes of nocardiosis. Despite utilization of protocol bronchoscopies with cultures for Nocardia, this organism remains a source of clinical complexity in the lung transplant population.

Pros and cons of automatic milking in Europe.

During the last several decades, new milking management systems have been introduced, of which development of automatic milking (AM) systems is a significant step forward. In Europe, AM has become an established management system and has shown to be much more than milking management. Factors such as milking, milk quality, feeding, cow traffic, grazing, and animal behavior are essential elements of AM. This system offers possibilities for more frequent milking and can be adapted to lactational stage. Increased milk yield with AM has been observed, but lack of increased production has also been reported from the field, probably due to less attention paid to the total management system. The AM system provides consistent milking routines, with those for teat stimulation and feeding during milking giving an adequate oxytocin release and milk ejection. Initially, reduced milk quality, such as increased FFA, total bacteria count, and somatic cell count (SCC), was observed. Increased FFA could be due to increased milking frequency or handling of the milk, although this has not yet been determined. The elevated total bacteria count was probably due to mismanagement because later studies indicated that teat cleaning in AM is sufficient to reduce spores and dirt on the teats. Significant positive effects on udder health and teat treatment were observed in some studies, possibly as an effect of quarter milking, a procedure whereby an individual teat cup is detached when milk flow is below the preset level for detachment. Well-functioning cow traffic is a prerequisite for successful AM system performance to obtain an optimal number of visits to the feeding area and the milking parlor for all cows. Technical stoppages in the AM system (i.e., the milking unit) increased milk SCC, and the variation and length of the milking interval seem to contribute to elevated SCC. Grazing is a common management routine in many countries. Different ways to motivate the cows to visit the milking parlor, such as shorter distance between barn and pasture, supplement feeding, access to water, and use of acoustic signals, have been tested. It was concluded that use of AM and grazing systems together is possible as long as the distance from the milking parlor to pasture is short. With proper management routines, it is possible to achieve a production level and animal well-being in AM systems that are at least as good as in conventional milking systems.

Quarter milking for improved detection of increased SCC.

The aim of the present study was to investigate whether milk composition and milk yield are changed in relation to a moderate increase in milk somatic cell count (SCC) in separate udder quarters. During a period of 13 weeks, 4158 bulk quarter milk samples from 68 cows were collected and analysed for milk SCC and milk composition. The sampling was done twice weekly. The cows were in different stages of lactation and in different lactation numbers. For calculations, three groups of cows were formed according to their SCC value. Group 1 cows, where all quarters had an SCC <100,000 cells/ml at all sampling occasions, were considered to be non-affected. Group 2 cows had one udder quarter with an increased SCC >100,000 cells/ml and 1.5-fold higher than the opposite quarter at one sampling occasion. For group 3 cows, the increase in SCC remained for several consecutive sampling occasions. Data from group 1 cows revealed that front and rear quarters were similar when compared with each other. For group 3 cows, the lactose content in milk decreased significantly, simultaneously with the increase in SCC and remained decreased for two sampling occasions after the initial increase in SCC. It was concluded that deviations in lactose content within front and rear quarters, respectively, may be a useful tool for detection of moderately increased SCC in separate udder quarters.

Family 7 cellobiohydrolases from Phanerochaete chrysosporium: crystal structure of the catalytic module of Cel7D (CBH58) at 1.32 A resolution and homology models of the isozymes.

Cellobiohydrolase 58 (Cel7D) is the major cellulase produced by the white-rot fungus Phanerochaete chrysosporium, constituting approximately 10 % of the total secreted protein in liquid culture on cellulose. The enzyme is classified into family 7 of the glycosyl hydrolases, together with cellobiohydrolase I (Cel7A) and endoglucanase I (Cel7B) from Trichoderma reesei. Like those enzymes, it catalyses cellulose hydrolysis with net retention of the anomeric carbon configuration. The structure of the catalytic module (431 residues) of Cel7D was determined at 3.0 A resolution using the structure of Cel7A from T. reesei as a search model in molecular replacement, and ultimately refined at 1.32 A resolution. The core structure is a beta-sandwich composed of two large and mainly antiparallel beta-sheets packed onto each other. A long cellulose-binding groove is formed by loops on one face of the sandwich. The catalytic residues are conserved and the mechanism is expected to be the same as for other family members. The Phanerochaete Cel7D binding site is more open than that of the T. reesei cellobiohydrolase, as a result of deletions and other changes in the loop regions, which may explain observed differences in catalytic properties. The binding site is not, however, as open as the groove of the corresponding endoglucanase. A tyrosine residue at the entrance of the tunnel may be part of an additional subsite not present in the T. reesei cellobiohydrolase. The Cel7D structure was used to model the products of the five other family 7 genes found in P. chrysosporium. The results suggest that at least two of these will have differences in specificity and possibly catalytic mechanism, thus offering some explanation for the presence of Cel7 isozymes in this species, which are differentially expressed in response to various growth conditions.

Kinetics of the coupled reaction catalysed by a fusion protein of beta-galactosidase and galactose dehydrogenase.

The mechanistic implications of the kinetic behaviour of a fusion protein of beta-galactosidase and galactose dehydrogenase have been analysed in view of predictions based on experimentally determined kinetic parameter values for the galactosidase and dehydrogenase activities of the protein. The results show that the time course of galactonolactone formation from lactose in the coupled reaction catalysed by the fusion protein can be most satisfactorily accounted for in terms of a free-diffusion mechanism when consideration is given to the mutarotation of the reaction intermediate galactose. It is concluded that no tenable kinetic evidence is available to support the proposal that the fusion protein catalyses galactonolactone formation from lactose by a mechanism involving channelling of galactose.

Mechanism of substrate inhibition in cellulose synergistic degradation.

A comprehensive experimental study of substrate inhibition in cellulose hydrolysis based on a well defined system is presented. The hydrolysis of bacterial cellulose by synergistically operating binary mixtures of cellobiohydrolase I from Trichoderma reesei and five different endoglucanases as well as their catalytic domains displays a characteristic substrate inhibition. This inhibition phenomenon is shown to require the two-domain structure of an intact cellobiohydrolase. The experimental data were in accordance with a mechanism where cellobiohydrolases previously bound to the cellulose by means of their cellulose binding domains are able to find chain ends by lateral diffusion. An increased substrate concentration at a fixed enzyme load will also increase the average diffusion distance/time needed for cellobiohydrolases to reach new chain ends created by endoglucanases, resulting in an apparent substrate inhibition of the synergistic action. The connection between the binding properties and the substrate inhibition is encouraging with respect to molecular engineering of the binding domain for optimal performance in biotechnological processes.

Double valve endocarditis.

BACKGROUND: There are little data concerning surgical outcomes in patients with native valve endocarditis affecting both the aortic and mitral valves. METHODS: From 1977 to 1998, 54 patients had simultaneous aortic and mitral valve grafting for native valve endocarditis. In 78%, mitral valve involvement was limited to the anterior leaflet, suggesting a jet lesion from the aortic valve. Surgical strategies included 31 valve repairs and valve replacement with mechanical (34), bioprosthetic (34), or allograft (9) prostheses. Three hundred twenty-five patient-years of follow-up were available for analysis (mean 6.0 +/- 4.8 years). RESULTS: There were no hospital deaths. Ten-year survival was 73%. Ten-year freedom from recurrent endocarditis was 84%, with risk peaking at 3 months, followed by a constant risk of 1.3%/yr. Choice of valvar procedure did not influence mortality or reinfection risk. CONCLUSIONS: The most common pattern of double valve infection was a jet lesion on the anterior mitral leaflet. Surgical treatment has late survival and freedom from reinfection similar to those of patients with single heart valve infection.

Tracheal cancer in Denmark: a nationwide study.

OBJECTIVE: Most published series on tracheal cancer reflect single institution experiences. We used the nationwide Danish Cancer Registry to report on characteristics and treatment of tracheal cancers in Denmark. METHODS: One hundred and nine cases of primary tracheal cancers were extracted from the registry in the period 1978-1995. The clinical data, histological distribution and treatment modalities were analyzed. The cancers were staged in four groups (stage I-IV) according to size, location and spread. RESULTS: Seventeen cases were diagnosed at autopsy. Ninety-two cases were diagnosed in vivo and 84% of these within 3 months after the first consultation. Sixty-three percent of the cancers were squamous cell carcinomas and only 7% were adenoid cystic carcinomas. The disease was at stage I in 21%, stage II in 23%, stage III in 6% and stage IV in 50%. The majority of the patients received radiotherapy as single treatment. Only nine patients were offered surgery (six were resected and three were found inoperable). The overall survival rates for cases diagnosed in vivo were 1-year 32%, 2-year 20% and 5-year 13%. For the resected patients the 5- and 15-year survival rates were 50%. CONCLUSIONS: Tracheal cancers were rare and adenoid cystic carcinomas not as frequent as generally believed. Surgery was rarely offered. A resectability rate of only 10% is not adequately explained by selection bias and indicates a nihilistic attitude based on ignorance about surgical treatment of tracheal cancers. A more dedicated and aggressive approach with centralized workup and radical treatment is strongly recommended.

An amperometric cellobiose dehydrogenase-based biosensor can be used for measurement of cellulase activity.

The hemoflavoenzyme cellobiose dehydrogenase (CDH, EC 1.1.99.18) from Phanerochaete chrysosporium has been used in an amperometric redox polymer-based biosensor. Used in conjugation with a FIA system this biosensor can replace colorimetric assays for measuring cellobiose liberated from cellulose in a series of cellulase-containing samples. The biosensor gave the same result as the Somogyi-Nelson method in a less time-consuming and laborious manner. The two methods showed about the same precision.

Structural basis for enantiomer binding and separation of a common beta-blocker: crystal structure of cellobiohydrolase Cel7A with bound (S)-propranolol at 1.9 A resolution.

Cellobiohydrolase Cel7A (previously called CBH 1), the major cellulase produced by the mould fungus Trichoderma reesei, has been successfully exploited as a chiral selector for separation of stereo-isomers of some important pharmaceutical compounds, e.g. adrenergic beta-blockers. Previous investigations, including experiments with catalytically deficient mutants of Cel7A, point unanimously to the active site as being responsible for discrimination of enantiomers. In this work the structural basis for enantioselectivity of basic drugs by Cel7A has been studied by X-ray crystallography. The catalytic domain of Cel7A was co-crystallised with the (S)-enantiomer of a common beta-blocker, propranolol, at pH 7, and the structure of the complex was determined and refined at 1. 9 A resolution. Indeed, (S)-propranolol binds at the active site, in glucosyl-binding subsites -1/+1. The catalytic residues Glu212 and Glu217 make tight salt links with the secondary amino group of (S)-propranolol. The oxygen atom attached to the chiral centre of (S)-propranolol forms hydrogen bonds to the nucleophile Glu212 O(epsilon1) and to Gln175 N(epsilon2), whereas the aromatic naphthyl moiety stacks with the indole ring of Trp376 in site +1. The bidentate charge interaction with the catalytic glutamate residues is apparently crucial, since no enantioselectivity has been obtained with the catalytically deficient mutants E212Q and E217Q. Activity inhibition experiments with wild-type Cel7A were performed in conditions close to those used for crystallisation. Competitive inhibition constants for (R)- and (S)-propranolol were determined at 220 microM and 44 microM, respectively, corresponding to binding free energies of 20 kJ/mol and 24 kJ/mol, respectively. The K(i) value for (R)-propranolol was 57-fold lower than the highest concentration, 12.5 mM, used in co-crystallisation experiments. Still several attempts to obtain a complex with the (R)-enantiomer have failed. By using cellobiose as a selective competing ligand, the retention of the enantiomers of propranolol on the chiral stationary phase (CSP) based on Cel7A mutant D214N were resolved into enantioselective and non- selective binding. The enantioselective binding was weaker for both enantiomers on D214N-CSP than on wild-type-CSP.