Pushing and pulling proteins into the yeast secretory pathway enhances recombinant protein secretion.

Yeasts and especially Pichia pastoris (syn Komagataella spp.) are popular microbial expression systems for the production of recombinant proteins. One of the key advantages of yeast host systems is their ability to secrete the recombinant protein into the culture media. However, secretion of some recombinant proteins is less efficient. These proteins include antibody fragments such as Fabs or scFvs. We have recently identified translocation of nascent Fab fragments from the cytosol into the endoplasmic reticulum (ER) as one major bottleneck. Conceptually, this bottleneck requires engineering to increase the flux of recombinant proteins at the translocation step by pushing on the cytosolic side and pulling on the ER side. This engineering strategy is well-known in the field of metabolic engineering. To apply the push-and-pull strategy to recombinant protein secretion, we chose to modulate the cytosolic and ER Hsp70 cycles, which have a key impact on the translocation process. After identifying the relevant candidate factors of the Hsp70 cycles, we combined the push-and-pull factors in a single strain and achieved synergistic effects for antibody fragment secretion. With this concept we were able to successfully engineer strains and improve protein secretion up to 5-fold for different model protein classes. Overall, titers of more than 1.3 g/L Fab and scFv were reached in bioreactor cultivations.

Patient reported outcome and cosmetic evaluation following implant-based breast-reconstruction with a titanized polypropylene mesh (TiLOOP® Bra): A prospective clinical study in 269 patients.

INTRODUCTION: Implant-based or expander-supported breast reconstruction is an established surgical method after mastectomies due to cancer or to prophylactic reasons. Patient reported outcome (PRO) and cosmetic outcome after breast reconstruction with a synthetic surgical mesh was investigated in a prospective, single-arm, multi-center study. MATERIAL AND METHODS: Primary or secondary implant-based breast reconstruction with support of TiLOOP® Bra was performed in 269 patients during the PRO-BRA study. PRO 12 months after breast reconstruction was evaluated using Breast-Q questionnaire. Cosmetic outcome was evaluated by two independent experts by means of pictures taken preoperatively and at the follow-up visits. RESULTS: Breast-Q and 12 months FU were completed by 210 women. Patients without adverse event had a significantly higher Breast-Q score for "sexual well-being" (p = 0.001); "psychosocial well-being" was negatively influenced by prior therapies (p < 0.01), and older patients had significantly lower scores at 12 months FU compared to pre-OP for "satisfaction with breasts" (p < 0.01) while the opposite was true for younger patients. Unilateral surgery resulted in reduced "satisfaction with breast" at 12 months FU (p < 0.01). Radiotherapy negatively influenced "satisfaction with breast", "sexual well-being" and "physical well-being chest". The cosmetic evaluation showed a significant difference (p < 0.001) in the evaluation by the patients and experts with the patients' assessment being worse compared to experts' assessment. CONCLUSION: Our study showed that two years after implant-based breast reconstruction with support of TiLOOP® Bra PRO is influenced by different factors. This information can be used to improve the decision-making process for women who chose implant-based breast reconstruction.

Gynecomastia - Conservative and Surgical Management.

Gynecomastia is defined as a unilateral or bilateral persistent benign mammary gland enlargement in men. Prevalence of asymptomatic gynecomastia is up to 65%. True gynecomastia must be distinguished from pseudogynecomastia. Typically, in true gynecomastia, a solid tissue mass is palpable below the nipple-areolar complex. Malignant changes such as male mammary carcinoma must always be ruled out. The causes of gynecomastia are diverse. An imbalance of female to male hormones triggers the onset of the disease. This imbalance can be caused by endogenous diseases like hyperthyroidism, chronic liver disease, primary or secondary gonadal failure, androgen resistance syndromes, medication, and drug abuse. A series of heart or hypertension medications can also trigger gynecomastia. A basic requirement of proper therapy planning is knowledge of the triggers and possible drug therapy options. Inquiring about the patient's lifestyle and medication history is essential. Drug therapy with tamoxifen may be considered at an early stage. For gynecomastia persisting over 12 months, surgical excision is the treatment of choice, and there are several surgical options available depending on the grade of the gynecomastia. A thoughtful approach to managing this condition can lead to high patient satisfaction.

GoldenPiCS: a Golden Gate-derived modular cloning system for applied synthetic biology in the yeast Pichia pastoris.

BACKGROUND: State-of-the-art strain engineering techniques for the host Pichia pastoris (syn. Komagataella spp.) include overexpression of homologous and heterologous genes, and deletion of host genes. For metabolic and cell engineering purposes the simultaneous overexpression of more than one gene would often be required. Very recently, Golden Gate based libraries were adapted to optimize single expression cassettes for recombinant proteins in P. pastoris. However, an efficient toolbox allowing the overexpression of multiple genes at once was not available for P. pastoris. METHODS: With the GoldenPiCS system, we provide a flexible modular system for advanced strain engineering in P. pastoris based on Golden Gate cloning. For this purpose, we established a wide variety of standardized genetic parts (20 promoters of different strength, 10 transcription terminators, 4 genome integration loci, 4 resistance marker cassettes). RESULTS: All genetic parts were characterized based on their expression strength measured by eGFP as reporter in up to four production-relevant conditions. The promoters, which are either constitutive or regulatable, cover a broad range of expression strengths in their active conditions (2-192% of the glyceraldehyde-3-phosphate dehydrogenase promoter P GAP ), while all transcription terminators and genome integration loci led to equally high expression strength. These modular genetic parts can be readily combined in versatile order, as exemplified for the simultaneous expression of Cas9 and one or more guide-RNA expression units. Importantly, for constructing multigene constructs (vectors with more than two expression units) it is not only essential to balance the expression of the individual genes, but also to avoid repetitive homologous sequences which were otherwise shown to trigger "loop-out" of vector DNA from the P. pastoris genome. CONCLUSIONS: GoldenPiCS, a modular Golden Gate-derived P. pastoris cloning system, is very flexible and efficient and can be used for strain engineering of P. pastoris to accomplish pathway expression, protein production or other applications where the integration of various DNA products is required. It allows for the assembly of up to eight expression units on one plasmid with the ability to use different characterized promoters and terminators for each expression unit. GoldenPiCS vectors are available at Addgene.

Cosmetic changes following surgery and accelerated partial breast irradiation using HDR interstitial brachytherapy : Evaluation by a multidisciplinary/multigender committee.

BACKGROUND: Patients with early-stage breast cancer can benefit from adjuvant accelerated partial breast irradiation (APBI) following breast-conserving surgery (BCS). This work reports on cosmetic results following APBI using multicatheter high-dose-rate interstitial brachytherapy (HDR-IBT). PATIENTS AND METHODS: Between 2006 and 2014, 114 patients received adjuvant APBI using multicatheter HDR-IBT. For each patient, two photographs were analyzed: the first was taken after surgery (baseline image) and the second at the last follow-up visit. Cosmesis was assessed by a multigender multidisciplinary team using the Harvard Breast Cosmesis Scale. Dose-volume histogram (DVH) parameters and the observed cosmetic results were investigated for potential correlations. RESULTS: The median follow-up period was 3.5 years (range 0.6-8.5 years). The final cosmetic scores were 30% excellent, 52% good, 14.5% fair, and 3.5% poor. Comparing the baseline and follow-up photographs, 59.6% of patients had the same score, 36% had a better final score, and 4.4% had a worse final score. Only lower target dose nonuniformity ratio (DNR) values (0.3 vs. 0.26; p = 0.009) were significantly associated with improved cosmetic outcome vs. same/worse cosmesis. CONCLUSION: APBI using multicatheter HDR-IBT adjuvant to BCS results in favorable final cosmesis. Deterioration in breast cosmesis occurs in less than 5% of patients. The final breast cosmetic outcome in patients treated with BCS and APBI using multicatheter HDR-IBT is influenced primarily by the cosmetic result of the surgery. A lower DNR value is significantly associated with a better cosmetic outcome.

New objective method in reporting the breast cosmesis after breast-conservative treatment based on nonstandardized photographs: The Objective Breast Cosmesis Scale.

PURPOSE: Subjective scoring is the most widely used approach in reporting the cosmetic outcome after breast-conservative therapy. This work introduces an objective system to document the breast cosmetic changes using nonstandardized photographs without scale calibration. METHODS AND MATERIALS: Two hundred twenty-eight photographs of 114 breast cancer patients were analyzed. Baseline photographs were taken after breast-conservation surgery and before partial breast irradiation. Further photographs were taken during followup. The photographs were taken with a frontal view of the patient and without any skin marks for scaling. The baseline and the last followup photographs were analyzed by measuring certain anatomic distances (representing the nipple displacement and the asymmetry in breast dimensions and contour) to calculate the objective breast cosmesis score (OBCS). The measurements represent the nipple displacement and the asymmetry in breast dimensions and contour. Same photographs were scored subjectively by a multidisciplinary team (MDT) using the Harvard breast cosmesis scale. The patient-reported self-scoring was also recorded. RESULTS: The MDT results were favorable (excellent∖good) in 72.3% of the photographs and adverse in 27.7%. Agreement among the MDT members was strong (intraclass correlation coefficient = 0.798, p < 0.001, 95% CI: 0.753-0.937, Cronbach's alpha = 0.809). The patient self-scoring was satisfactory in 82.5% of the cases and nonsatisfactory in 17.5%. The results of the OBCS ranged between 0.0 and 20.4 with a median value of 4.5. There was a strong significant correlation between the OBCS and both the MDT subjective scoring (p < 0.001) and the patient self-scoring (p < 0.001). CONCLUSIONS: The OBCS seems to be eligible for the objective assessment of cosmesis after breast-conservative therapy using nonstandardized photographs without scale calibration.

The prognostic impact of age in different molecular subtypes of breast cancer.

Breast cancer is a heterogeneous entity composed of distinct molecular subgroups with different molecular and clinical features. We analyzed the association between molecular breast cancer subgroups, age at diagnosis, and prognosis in a compilation of publicly available gene expression datasets. Affymetrix gene expression data (U133A or U133Plus2.0 arrays) of 4467 breast cancers from 40 datasets were compiled and homogenized. Breast cancer subgroups were defined based on expression of ESR1, PR, HER2, and Ki67. Event-free survival was calculated as recurrence-free survival or distant metastasis-free survival if recurrence-free survival was not available. Young age at diagnosis is associated with higher frequency of triple negative and HER2 subtypes and lower frequency of luminal A breast cancers. The 5-year event-free survival rates of patients aged less than 40, between 40 and 50, and >50 years were 54.3 ± 3.5, 68.5 ± 1.9, and 70.4 ± 1.3 %, respectively. When controlling for breast cancer subtype, we found that age <40 years remained significantly associated with poor prognosis in triple negative breast cancer. The effect was modest in luminal tumors and not found in HER2 subtype. Both subtypes and age retained their significances in multivariate analysis. Association of age at diagnosis with molecular breast cancer subtype contributes to its important role as prognostic factor among patients with breast cancer. Still, within the group of triple negative breast cancer, young age <40 years has a significant prognostic value which was retained in multivariate analysis.

Intraoperative assessment of margins in breast conservative surgery--still in use?

A positive margin in breast conserving surgery is associated with an increased risk of local recurrence. Failure to achieve clear margins results in re-excision procedures. Methods for intraoperative assessment of margins have been developed, such as frozen section analysis, touch preparation cytology, near-infrared fluorescence optical imaging, x-ray diffraction technology, high-frequency ultrasound, micro-CT, and radiofrequency spectroscopy. In this article, options that might become the method of choice in the future are discussed.

MarginProbe®--final results of the German post-market study in breast conserving surgery of ductal carcinoma in situ.

The goal in breast conserving surgery (BCS) of ductal carcinoma in situ (DCIS) is removal of the tumor with a clear surgical margin. However, re-excision rates are regrettably high. To date, there are no adequate procedures for intraoperative margin assessment of DCIS. A multicenter, single arm study was conducted to evaluate the benefit of a novel device (MarginProbe®) in intraoperative margin assessment during BCS of DCIS, the associated reduction of re-excisions and the cosmetic outcome of the treated patients. We present results of 42 patients enrolled in 3 German institutions. The device was used as an adjunctive tool to standard of care. The device use was associated with a reduction in re-excision rates by 56%, from 39% to 17% (p = 0.018).

Guideline conformity treatment in young women with early-onset breast cancer in Germany.

BACKGROUND: The purpose of the study was to characterize the treatment of young mothers with breast cancer in Germany and to investigate whether it followed applicable guidelines. METHOD: Retrospective data obtained between 2002 and 2011 for 1,053 mothers with children of < 12 years of age with adjuvantly treated loco-regional primary breast cancer were analyzed. Collected data included sociodemographic data, TNM stage, biology of tumor and therapies. Actually received therapies were compared to those suggested in guideline treatment plans. RESULTS: The mean age of the patients was 39 years. 97% of the women with node positivity received an axillary dissection. Overall, 90% of the patients received chemotherapy with a guideline adherence range of 87-99% depending on clinical parameters. For radiation therapy, guideline adherence was high (range 82-100%). 95% of the patients with a hormone receptor-positive tumor received endocrine therapy; in 94%, tamoxifen therapy was performed in compliance with guidelines, whereas gonadotropin-releasing hormone (GnRH) agonist therapy complied with the guidelines in 52% of the cases. CONCLUSION: Guideline adherence in young mothers with breast cancer in Germany was high (with the exception of GnRH therapy), as comorbidity or the ambiguity of the therapeutic success does not need to be considered as much in this young, otherwise usually healthy, cohort compared to an age-heterogeneous group.

Maternal factor V Leiden and prothrombin mutations do not seem to contribute to the occurrence of two or more than two consecutive miscarriages in Caucasian patients.

BACKGROUND: We analysed the prevalence of the most common hereditary thrombophilia (hTP) - factor V Leiden (FVL) mutation, prothrombin 20210 G>A substitution (PT) - and the 677 C>T replacement in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene in Caucasian patients with a history of two and more consecutive recurrent miscarriages (RMs) as compared to healthy controls with an identical ethnic background and at least one live birth. METHODS: A multicenter analysis of three hTP was performed in 641 RM patients identically screened at specialized university centres. RESULTS: The study groups consisted of 240 patients with 2 (1) and 401 patients with >2 miscarriages (2) and were compared with 157 controls. There was no significant difference in the prevalence of the hTP between RM patients and controls nor within the two study groups. Subgroup analysis showed that the homozygous MTHFR polymorphism was significantly more prevalent in the study group 2 as compared to study group 1 (13.9 versus 7.9%, P = 0.02). CONCLUSION: In Caucasians, maternal FVL or PT mutations do not seem to contribute to the pathophysiology of RM, irrespective of the number of miscarriages. However, the role of the homozygous MTHFR polymorphism merits further investigation.

Reproducibility of fetal ductus venosus blood flow velocity waveforms during first stage of labor.

OBJECTIVES: Fetal ductus venosus (DV) blood flow velocity waveforms are significantly altered during contractions in first stage of labor. We have evaluated the reproducibility of these waveforms during and between contractions. METHODS: 90 women between 37th and 41st week of gestation were included in the study. The measurements of the DV were obtained by three different investigators during and between contractions. The pulsatility index for veins (PIV), peak velocity index for veins (PVIV), and fetal heart rate (FHR) were calculated offline. Furthermore, differences in PVIV (PVIVdiff) and PIV (PIVdiff) and a mean of FHR (FHRmean) were calculated. Statistical analysis was used to verify differences in the PIV, PVIV and FHR during and between contractions of each subgroup as well as for the PVIVdiff, PIVdiff and FHRmean between the subgroups. RESULTS: Investigator 1 examined 49 patients (group 1); investigator 2, 17 patients (group 2) and investigator 3, 24 patients (group 3), respectively. In all subgroups, there was a significant difference for the PVIV and PIV during and between contractions, but not for FHR. There was a correlation between gestational age and PVIVdiff, PIVdiff and FHRmean as well as for maternal age and PVIVdiff and PIVdiff. PVIVdiff and PIVdiff showed significant differences in group 1 compared to groups 2 and 3 (p < 0.001), but not between groups 2 and 3. CONCLUSIONS: Significant differences in the measurements in the three subgroups have been revealed, which may be due to different timing of measurement during the contraction. For future studies further standardization of the measurement protocol should be established.

New technologies in breast cancer surgery.

Since breast-conserving surgery has become the gold standard for early breast cancer, the development of less radical or less burdensome technologies has been pressed for in order to preserve the patient from unnecessary harm through the operative procedure. Different technical approaches are under evaluation, and some of them are already being used in the clinical setting. The aim of this article is to present a perspective on future breast cancer surgery by shedding light on the current innovative and new techniques.

Protein trafficking, ergosterol biosynthesis and membrane physics impact recombinant protein secretion in Pichia pastoris.

BACKGROUND: The increasing availability of 'omics' databases provide important platforms for yeast engineering strategies since they offer a lot of information on the physiology of the cells under diverse growth conditions, including environmental stresses. Notably, only a few of these approaches have considered a performance under recombinant protein production conditions. Recently, we have identified a beneficial effect of low oxygen availability on the expression of a human Fab fragment in Pichia pastoris. Transcriptional analysis and data mining allowed for the selection of potential targets for strain improvement. A first selection of these candidates has been evaluated as recombinant protein secretion enhancers. RESULTS: Based on previous transcriptomics analyses, we selected 8 genes for co-expression in the P. pastoris strain already secreting a recombinant Fab fragment. Notably, WSC4 (which is involved in trafficking through the ER) has been identified as a novel potential target gene for strain improvement, with up to a 1.2-fold increase of product yield in shake flask cultures. A further transcriptomics-based strategy to modify the yeast secretion system was focused on the ergosterol pathway, an aerobic process strongly affected by oxygen depletion. By specifically partially inhibiting ergosterol synthesis with the antifungal agent fluconazole (inhibiting Erg11p), we tried to mimic the hypoxic conditions, in which the cellular ergosterol content was significantly decreased. This strategy led to an improved Fab yield (2-fold) without impairing cellular growth. Since ergosterol shortage provokes alterations in the plasma membrane composition, an important role of this cellular structure in protein secretion is suggested. This hypothesis was additionally supported by the fact that the addition of non-ionic surfactants also enhanced Fab secretion. CONCLUSIONS: The current study presents a systems biotechnology-based strategy for the engineering of the industrially important yeast P. pastoris combining the use of host specific DNA microarray technologies and physiological studies under well defined environmental conditions. Such studies allowed for the identification of novel targets related with protein trafficking and ergosterol biosynthesis for improved recombinant protein production. Nevertheless, further studies will be required to elucidate the precise mechanisms whereby membrane biogenesis and composition impact on protein secretion in P. pastoris.

The impact of oxygen on the transcriptome of recombinant S. cerevisiae and P. pastoris - a comparative analysis.

BACKGROUND: Saccharomyces cerevisiae and Pichia pastoris are two of the most relevant microbial eukaryotic platforms for the production of recombinant proteins. Their known genome sequences enabled several transcriptomic profiling studies under many different environmental conditions, thus mimicking not only perturbations and adaptations which occur in their natural surroundings, but also in industrial processes. Notably, the majority of such transcriptome analyses were performed using non-engineered strains.In this comparative study, the gene expression profiles of S. cerevisiae and P. pastoris, a Crabtree positive and Crabtree negative yeast, respectively, were analyzed for three different oxygenation conditions (normoxic, oxygen-limited and hypoxic) under recombinant protein producing conditions in chemostat cultivations. RESULTS: The major differences in the transcriptomes of S. cerevisiae and P. pastoris were observed between hypoxic and normoxic conditions, where the availability of oxygen strongly affected ergosterol biosynthesis, central carbon metabolism and stress responses, particularly the unfolded protein response. Steady state conditions under low oxygen set-points seemed to perturb the transcriptome of S. cerevisiae to a much lesser extent than the one of P. pastoris, reflecting the major tolerance of the baker's yeast towards oxygen limitation, and a higher fermentative capacity. Further important differences were related to Fab production, which was not significantly affected by oxygen availability in S. cerevisiae, while a clear productivity increase had been previously reported for hypoxically grown P. pastoris. CONCLUSIONS: The effect of three different levels of oxygen availability on the physiology of P. pastoris and S. cerevisiae revealed a very distinct remodelling of the transcriptional program, leading to novel insights into the different adaptive responses of Crabtree negative and positive yeasts to oxygen availability. Moreover, the application of such comparative genomic studies to recombinant hosts grown in different environments might lead to the identification of key factors for efficient protein production.

Coexistent parathyroid adenoma and thyroid papillary carcinoma in pregnancy.

BACKGROUND: Primary hyperparathyroidism in pregnant women is a rare antenatal complication. Maternal symptoms include nephrolithiasis, bone disease, pancreatitis, hyperemesis, muscle weakness, mental status changes, and hypercalcemic crisis. Fetal complications comprise intrauterine growth retardation, low birth weight, preterm delivery, intrauterine fetal death, postpartum neonatal titanic crisis, and permanent hypoparathyroidism. CASE: A 23-year-old gravida was referred to our clinic with severe hyperemesis and weight loss at 32 gestational weeks. She was diagnosed with primary hyperparathyroidism because of parathyroid adenoma and treated with surgery where a synchronous thyroid papillary carcinoma was detected. After right parathyroidectomy and right subtotal lobectomy of the thyroid, the patient was stable and laboratory and clinical findings normalized. The fetus' state was monitored via reassuring non-stress cardiotocography and repeated sonographic exams until birth. Delivery was induced at 35 weeks of gestation because of preeclampsia. CONCLUSION: Parathyroid adenoma in pregnancy is a rare maternal condition with potential impact on the advancing pregnancy. Generally, a surgical approach is recommended with thorough inspection of the thyroid gland so that any irregular structure might be removed during the same surgical intervention for diagnostic reasons. In this reported case, a coexistent papillary carcinoma of the thyroid was detected accidentally and removed successfully.

Influence of growth temperature on the production of antibody Fab fragments in different microbes: a host comparative analysis.

Microorganisms encounter diverse stress conditions in their native habitats but also during fermentation processes, which have an impact on industrial process performance. These environmental stresses and the physiological reactions they trigger, including changes in the protein folding/secretion machinery, are highly interrelated. Thus, the investigation of environmental factors, which influence protein expression and secretion is still of great importance. Among all the possible stresses, temperature appears particularly important for bioreactor cultivation of recombinant hosts, as reductions of growth temperature have been reported to increase recombinant protein production in various host organisms. Therefore, the impact of temperature on the secretion of proteins with therapeutic interest, exemplified by a model antibody Fab fragment, was analyzed in five different microbial protein production hosts growing under steady-state conditions in carbon-limited chemostat cultivations. Secretory expression of the heterodimeric antibody Fab fragment was successful in all five microbial host systems, namely Saccharomyces cerevisiae, Pichia pastoris, Trichoderma reesei, Escherichia coli and Pseudoalteromonas haloplanktis. In this comparative analysis we show that a reduction of cultivation temperature during growth at constant growth rate had a positive effect on Fab 3H6 production in three of four analyzed microorganisms, indicating common physiological responses, which favor recombinant protein production in prokaryotic as well as eukaryotic microbes.

A multi-level study of recombinant Pichia pastoris in different oxygen conditions.

BACKGROUND: Yeasts are attractive expression platforms for many recombinant proteins, and there is evidence for an important interrelation between the protein secretion machinery and environmental stresses. While adaptive responses to such stresses are extensively studied in Saccharomyces cerevisiae, little is known about their impact on the physiology of Pichia pastoris. We have recently reported a beneficial effect of hypoxia on recombinant Fab secretion in P. pastoris chemostat cultivations. As a consequence, a systems biology approach was used to comprehensively identify cellular adaptations to low oxygen availability and the additional burden of protein production. Gene expression profiling was combined with proteomic analyses and the 13C isotope labelling based experimental determination of metabolic fluxes in the central carbon metabolism. RESULTS: The physiological adaptation of P. pastoris to hypoxia showed distinct traits in relation to the model yeast S. cerevisiae. There was a positive correlation between the transcriptomic, proteomic and metabolic fluxes adaptation of P. pastoris core metabolism to hypoxia, yielding clear evidence of a strong transcriptional regulation component of key pathways such as glycolysis, pentose phosphate pathway and TCA cycle. In addition, the adaptation to reduced oxygen revealed important changes in lipid metabolism, stress responses, as well as protein folding and trafficking. CONCLUSIONS: This systems level study helped to understand the physiological adaptations of cellular mechanisms to low oxygen availability in a recombinant P. pastoris strain. Remarkably, the integration of data from three different levels allowed for the identification of differences in the regulation of the core metabolism between P. pastoris and S. cerevisiae. Detailed comparative analysis of the transcriptomic data also led to new insights into the gene expression profiles of several cellular processes that are not only susceptible to low oxygen concentrations, but might also contribute to enhanced protein secretion.

Nulliparous patients with small stature delivering at term have an increased risk of secondary cesarean section.

PURPOSE: To evaluate the obstetric outcome of pregnant patients with small stature (<5th percentile) with regard to the mode of delivery, maternal injuries, and neonatal parameters. METHODS: Retrospective cohort analysis of 13 years of deliveries. Two groups: group A, patients with a height below the 5th percentile, and group B, patients with a body height between the 25th and 75th percentile. RESULTS: Patients with a body height between the 25th and 75th percentiles showed significantly more spontaneous vaginal deliveries. Secondary cesarean sections (CS) were significantly seen more often in mothers with a small body height. The fetal outcome did not differ significantly between both groups (APGAR, arterial cord pH, base excess). CONCLUSIONS: Patients with body height below the 5th percentile were found to have a significantly higher rate of secondary CS. As less than half of our patients with a body height below the 5th percentile were found to have delivered spontaneously at term, pregnancies in small patients should be recognized by obstetricians to be at a specific risk. Whereas the neonatal outcome appears to be comparable between nulliparous women with a body height below the 5th percentile and those with a body height between the 25th and 75th percentiles, small mothers carry a significantly elevated risk of surgical delivery, which should be addressed in prospective studies and in counseling these patients.

Macromolecular and elemental composition analysis and extracellular metabolite balances of Pichia pastoris growing at different oxygen levels.

BACKGROUND: Analysis of the cell operation at the metabolic level requires collecting data of different types and to determine their confidence level. In addition, the acquired information has to be combined in order to obtain a consistent operational view. In the case of Pichia pastoris, information of its biomass composition at macromolecular and elemental level is scarce particularly when different environmental conditions, such as oxygen availability or, genetic backgrounds (e.g. recombinant protein production vs. non production conditions) are compared. RESULTS: P. pastoris cells growing in carbon-limited chemostat cultures under different oxygenation conditions (% O2 in the bioreactor inlet gas: 21%, 11% and 8%, corresponding to normoxic, oxygen-limiting and hypoxic conditions, respectively), as well as under recombinant protein (antibody fragment, Fab) producing and non-producing conditions, were analyzed from different points of view. On the one hand, the macromolecular and elemental composition of the biomass was measured using different techniques at the different experimental conditions and proper reconciliation techniques were applied for gross error detection of the measured substrates and products conversion rates. On the other hand, fermentation data was analyzed applying elemental mass balances. This allowed detecting a previously missed by-product secreted under hypoxic conditions, identified as arabinitol (aka. arabitol). After identification of this C5 sugar alcohol as a fermentation by-product, the mass balances of the fermentation experiments were validated. CONCLUSIONS: After application of a range of analytical and statistical techniques, a consistent view of growth parameters and compositional data of P. pastoris cells growing under different oxygenation conditions was obtained. The obtained data provides a first view of the effects of oxygen limitation on the physiology of this microorganism, while recombinant Fab production seems to have little or no impact at this level of analysis. Furthermore, the results will be highly useful in other complementary quantitative studies of P. pastoris physiology, such as metabolic flux analysis.