Delayed microsurgical revascularization in an acute ischemic stroke based on perfusion study.

A 58-year-old patient presented with a severe neurological deficit due to a stroke caused by an occlusion of the left internal carotid artery siphon. Standard treatment failed and neurosurgical consult was delayed. Because of a favorable perfusion imaging finding, microsurgical revascularization via an extra-intracranial bypass (left superficial temporal artery - left middle cerebral artery) was performed 36 hours after the onset of the symptoms. The outcome of the patient was favorable. The authors want to emphasize the need to actively seek patients with a severe neurological deficit and still viable brain tissue. The time window and treatment alternatives are discussed.

Expression of circular RNAs in myelodysplastic neoplasms and their association with mutations in the splicing factor gene SF3B1.

Mutations in the splicing factor 3b subunit 1 (SF3B1) gene are frequent in myelodysplastic neoplasms (MDS). Because the splicing process is involved in the production of circular RNAs (circRNAs), we investigated the impact of SF3B1 mutations on circRNA processing. Using RNA sequencing, we measured circRNA expression in CD34+ bone marrow MDS cells. We defined circRNAs deregulated in a heterogeneous group of MDS patients and described increased circRNA formation in higher-risk MDS. We showed that the presence of SF3B1 mutations did not affect the global production of circRNAs; however, deregulation of specific circRNAs was observed. Particularly, we demonstrated that strong upregulation of circRNAs processed from the zinc finger E-box binding homeobox 1 (ZEB1) transcription factor; this upregulation was exclusive to SF3B1-mutated patients and was not observed in those with mutations in other splicing factors or other recurrently mutated genes, or with other clinical variables. Furthermore, we focused on the most upregulated ZEB1-circRNA, hsa_circ_0000228, and, by its knockdown, we demonstrated that its expression is related to mitochondrial activity. Using microRNA analyses, we proposed miR-1248 as a direct target of hsa_circ_0000228. To conclude, we demonstrated that mutated SF3B1 leads to deregulation of ZEB1-circRNAs, potentially contributing to the defects in mitochondrial metabolism observed in SF3B1-mutated MDS.

Tissue expression analysis of cervical mucus proteome.

Cervical mucus is a viscous fluid functioning as a cervix plug. Products of the endometrial and cervical glands can be detected in the cervical mucus. Cervical mucus is further enriched with transudate originating from the fallopian tubes and proteins originating from the ovaries, peritoneum and distant tissues. With increasing levels of ovarian estrogens, the properties of cervical mucus for possible collection and processing change appropriately. For these reasons, we chose a group of 10 patients treated in the center of assisted reproduction by controlled ovarian stimulation for in vitro fertilization. This study focuses on the proteomic characterization of cervical mucus and localizes the possible sources of the identified proteins. The most abundant proteins were extracellular proteins, mainly mucins; however, most of the identified proteins, present usually in lower quantities, were of intracellular origin. The tissue analysis revealed that proteins from female reproductive organs are also expressed in other tissues in addition to female reproductive organs, but also proteins specific to the testis, liver, placenta, retina, and cerebellum. This study confirms the suitability and high potential of cervical mucus as a source of proteomic bio-markers not only for the dia-gnosis of the female reproductive tract.

Proteome Mapping of Cervical Mucus and Its Potential as a Source of Biomarkers in Female Tract Disorders.

Cervical mucus (CM) is a viscous fluid that is produced by the cervical glands and functions as a uterine cervix plug. Its viscosity decreases during ovulation, providing a window for non-invasive sampling. This study focuses on proteomic characterization of CM to evaluate its potential as a non-invasively acquired source of biomarkers and in understanding of molecular (patho)physiology of the female genital tract. The first objective of this work was to optimize experimental workflow for CM processing and the second was to assess differences in the proteomic composition of CM during natural ovulatory cycles obtained from intrauterine insemination (IUI) cycles and in vitro fertilization (IVF) cycles with controlled ovarian hyperstimulation. Proteomic analysis of CM samples revealed 4370 proteins involved in processes including neutrophil degranulation, cellular stress responses, and hemostasis. Differential expression analysis revealed 199 proteins enriched in IUI samples and 422 enriched in IVF. The proteins enriched in IUI were involved in phosphatidic acid synthesis, responses to external stimulus, and neutrophil degranulation, while those enriched in IVF samples were linked to neutrophil degranulation, formation of a cornified envelope and hemostasis. Subsequent analyses clarified the protein composition of the CM and how it is altered by hormonal stimulation of the uterus.

Hormone receptor conversion in metastatic breast cancer.

Background/Objective: Hormone receptor (HR) status is one of the key factors in determining the treatment of breast cancer. Previous studies suggested that HR status may change in metastatic tissue. However, available studies focused mainly on primary biopsies and there are only few trials comparing HR status in the primary tumour and the metastasis using material from complete resection. The aim of the study was to determine the frequency of HR alterations in metastatic breast cancer. Materials and methods: The study retrospectively examines a total of 50 patients who underwent brain, lung, or liver metastasectomy for metastatic breast cancer between January 2000 and January 2019. Results: HR conversion was observed in a total of 30 cases (60.0%), while HER-2/neu (human epidermal growth factor receptor 2) discrepancy surprisingly occurred only in one case (2.0%). A change in immunophenotype occurred in 28% of cases. Triple-negativity was more frequent in brain metastases (p = 0.039). Conclusions: We have confirmed that HR conversion between the primary tumour and its metastases occurs in a significant number of cases, which has important implications for further treatment decisions.

Diurnal and seasonal differences in cardiopulmonary response to exercise in morning and evening chronotypes.

Circadian clocks regulate multiple physiological domains from molecular to behavioral levels and adjust bodily physiology to seasonal changes in day length. Circadian regulation of cellular bioenergy and immunity in the cardiovascular and muscle systems may underpin the individual diurnal differences in performance capacity during exercise. Several studies have shown diurnal differences in cardiopulmonary parameters at maximal and submaximal workloads in morning and evening circadian human phenotypes. However, the effect of seasons on these changes was not elucidated. In this study, we recruited subjects with Morningness-Eveningness Questionnaire scores corresponding to morning and evening types. Subjects underwent morning (7:00-9:00) and evening (20:00-22:00) maximal workload spiroergometry in both winter and summer seasons. We analyzed their performance time, anaerobic threshold, heart rate, and respiratory parameters. Our results suggest that evening types manifest diurnal variations in physical performance, particularly in winter. They also have slower heart rate recovery than morning types, irrespective of the time of day or season. Compared to winter, the chronotype effect on the magnitude of morning-evening differences in performance time, maximal heart rate, and anaerobic threshold onset was more significant in summer. Our data are in concordance with previous observations and confirm the difference between morning and evening types in the timing of maximum performance capacity.

Structural determinants for subnanomolar inhibition of the secreted aspartic protease Sapp1p from Candida parapsilosis.

Pathogenic Candida albicans yeasts frequently cause infections in hospitals. Antifungal drugs lose effectiveness due to other Candida species and resistance. New medications are thus required. Secreted aspartic protease of C. parapsilosis (Sapp1p) is a promising target. We have thus solved the crystal structures of Sapp1p complexed to four peptidomimetic inhibitors. Three potent inhibitors (Ki: 0.1, 0.4, 6.6 nM) resembled pepstatin A (Ki: 0.3 nM), a general aspartic protease inhibitor, in terms of their interactions with Sapp1p. However, the weaker inhibitor (Ki: 14.6 nM) formed fewer nonpolar contacts with Sapp1p, similarly to the smaller HIV protease inhibitor ritonavir (Ki: 1.9 µM), which, moreover, formed fewer H-bonds. The analyses have revealed the structural determinants of the subnanomolar inhibition of C. parapsilosis aspartic protease. Because of the high similarity between Saps from different Candida species, these results can further be used for the design of potent and specific Sap inhibitor-based antimycotic drugs.

Complications Following Decompressive Craniectomy.

BACKGROUND: Decompressive craniectomy (DC) has become the definitive surgical procedure to manage a medically intractable rise in intracranial pressure. DC is a life-saving procedure resulting in lower mortality but also higher rates of severe disability. Although technically straightforward, DC is accompanied by many complications. It has been reported that complications are associated with worse outcome. We reviewed a series of patients who underwent DC at our department to establish the incidence and types of complications. METHODS: We retrospectively evaluated the incidence of complications after DC performed in 135 patients during the time period from January 2013 to December 2018. Postoperative complications were evaluated using clinical status and CT during 6 months of follow-up. In addition, the impact of potential risk factors on the incidence of complications and the impact of complications on outcome were assessed. RESULTS: DC was performed in 135 patients, 93 of these for trauma, 22 for subarachnoid hemorrhage, 13 for malignant middle cerebral artery infarction, and 7 for intracerebral hemorrhage. Primary DC was performed in 120 patients and secondary DC in 15 patients. At least 1 complication occurred in each of 100 patients (74%), of which 22 patients (22%) were treated surgically. The following complications were found: edema or hematoma of the temporal muscle (34 times), extracerebral hematoma (33 times), extra-axial fluid collection (31 times), hemorrhagic progression of contusions (19 times), hydrocephalus (12 times), intraoperative malignant brain edema (10 times), temporal muscle atrophy (7 times), significant intraoperative blood loss (6 times), epileptic seizures (5 times), and skin necrosis (4 times). Trauma (p = 0.0006), coagulopathy (p = 0.0099), and primary DC (p = 0.0252) were identified as risk factors for complications. There was no significant impact of complications on outcome. CONCLUSIONS: The incidence of complications following DC is high. However, we did not confirm a significant impact of complications on outcome. We emphasize that some phenomena are so frequent that they can be considered a consequence of primary injury or natural sequelae of the DC rather than its direct complication.

Cellular Localization of Carbonic Anhydrase Nce103p in Candida albicans and Candida parapsilosis.

Pathogenic yeasts Candida albicans and Candida parapsilosis possess a ß-type carbonic anhydrase Nce103p, which is involved in CO2 hydration and signaling. C. albicans lacking Nce103p cannot survive in low CO2 concentrations, e.g., in atmospheric growth conditions. Candida carbonic anhydrases are orthologous to the Saccharomyces cerevisiae enzyme, which had originally been detected as a substrate of a non-classical export pathway. However, experimental evidence on localization of C. albicans and C. parapsilosis carbonic anhydrases has not been reported to date. Immunogold labeling and electron microscopy used in the present study showed that carbonic anhydrases are localized in the cell wall and plasmatic membrane of both Candida species. This localization was confirmed by Western blot and mass spectrometry analyses of isolated cell wall and plasma membrane fractions. Further analysis of C. albicans and C. parapsilosis subcellular fractions revealed presence of carbonic anhydrases also in the cytosolic and mitochondrial fractions of Candida cells cultivated in shaken liquid cultures, under the atmospheric conditions.

Functional Characterization of Secreted Aspartyl Proteases in Candida parapsilosis.

Candida parapsilosis is an emerging non-albicans Candida species that largely affects low-birth-weight infants and immunocompromised patients. Fungal pathogenesis is promoted by the dynamic expression of diverse virulence factors, with secreted proteolytic enzymes being linked to the establishment and progression of disease. Although secreted aspartyl proteases (Sap) are critical for Candida albicans pathogenicity, their role in C. parapsilosis is poorly elucidated. In the present study, we aimed to examine the contribution of C. parapsilosisSAPP genes SAPP1, SAPP2, and SAPP3 to the virulence of the species. Our results indicate that SAPP1 and SAPP2, but not SAPP3, influence adhesion, host cell damage, phagosome-lysosome maturation, phagocytosis, killing capacity, and cytokine secretion by human peripheral blood-derived macrophages. Purified Sapp1p and Sapp2p were also shown to efficiently cleave host complement component 3b (C3b) and C4b proteins and complement regulator factor H. Additionally, Sapp2p was able to cleave factor H-related protein 5 (FHR-5). Altogether, these data demonstrate the diverse, significant contributions that SAPP1 and SAPP2 make to the establishment and progression of disease by C. parapsilosis through enabling the attachment of the yeast cells to mammalian cells and modulating macrophage biology and disruption of the complement cascade.IMPORTANCE Aspartyl proteases are present in various organisms and, among virulent species, are considered major virulence factors. Host tissue and cell damage, hijacking of immune responses, and hiding from innate immune cells are the most common behaviors of fungal secreted proteases enabling pathogen survival and invasion. C. parapsilosis, an opportunistic human-pathogenic fungus mainly threatening low-birth weight neonates and children, possesses three SAPP protein-encoding genes that could contribute to the invasiveness of the species. Our results suggest that SAPP1 and SAPP2, but not SAPP3, influence host evasion by regulating cell damage, phagocytosis, phagosome-lysosome maturation, killing, and cytokine secretion. Furthermore, SAPP1 and SAPP2 also effectively contribute to complement evasion.

Patient Satisfaction with General versus Local Anesthesia during Carotid Endarterectomy.

BACKGROUND AND STUDY AIMS: Both general and local anesthesia are used in our department for carotid endarterectomy (CEA). The decision as to which anesthetic technique to use during surgery is made individually. The aim of our study was to evaluate patient satisfaction and preference with the anesthesia type used. MATERIAL AND METHODS: The satisfaction of a group of 205 patients with regard to anesthesia used and their future preferences were evaluated prospectively through a questionnaire. The reasons for dissatisfaction were assessed. RESULTS: CEA was performed under general anesthesia (GA) in 159 cases (77.6%) and under local anesthesia (LA) in 46 cases (22.4%). In the GA group, 148 patients (93.1%) were satisfied; 30 patients (65.2%) in the LA group were satisfied (p < 0.0001). The reason for dissatisfaction with GA were postoperative nausea and vomiting (7 patients), postoperative psychological alteration (3), and fear of GA (1). The reasons for dissatisfaction with LA were intraoperative pain (9 patients), intraoperative discomfort and stress (5), and intraoperative breathing problems (2). Of the GA group, 154 (96.9%) patients would prefer GA again, and of the LA group, 28 (60.9%) patients would prefer LA if operated on again (p < 0.0001). Overall, 172 patients (83.9%) would prefer GA in the future, and 33 patients (16.1%) would prefer LA. CONCLUSION: Overall patient satisfaction with CEA performed under both GA and LA is high. Nevertheless, in the GA group, patient satisfaction and future preference were significantly higher. Both GA and LA have advantages and disadvantages for CEA. An optimal approach is to make use of both anesthetic techniques based on their individual indications and patient preference.

Indications for General versus Local Anesthesia during Carotid Endarterectomy.

BACKGROUND AND STUDY AIMS: Both general anesthesia (GA) and local anesthesia (LA) are used in our department for carotid endarterectomy. The decision of which anesthetic technique to use during surgery is made on an individual basis. The aim of our study was to analyze the reasons for using GA or LA. MATERIAL AND METHODS: The reasons that led to the selection of either GA or LA were analyzed retrospectively in a group of 409 patients. RESULTS: GA was used in 304 patients (74%) and LA in 105 patients (26%). The reasons for a preference for GA were clopidogrel use (88 patients), patient preference (80), increased risk of shunt insertion (43), unfavorable anatomical conditions (41), surgeon preference (21), simultaneous carotid endarterectomy and cardiac surgery (18), emergent carotid endarterectomy (12), and sleep apnea syndrome (1). The reasons for selecting LA were internal comorbidities (46 patients), patient preference (39), unavailability of intraoperative electrophysiologic monitoring (15), and pacemaker (5). CONCLUSION: GA is the dominant choice for carotid endarterectomy in our department because of its prevailing benefits and its preference among neurosurgeons and patients. However, in some subgroups of patients, LA is preferable. An optimal approach is therefore an individual indication for both anesthesia techniques.

Decolorization and detoxification of textile wastewaters by recombinant Myceliophthora thermophila and Trametes trogii laccases.

Laccases are multi-copper oxidoreductases with broad biotechnological applications. Here, we report detailed biochemical characterization of purified recombinant laccases originating from Myceliophthora thermophila (MtL) and Trametes trogii (TtL). We identified optimal conditions for decolorization of commercial dyes and textile wastewater samples. We also tested the toxicity of decolorized wastewater samples using human peripheral blood mononuclear cells. MtL and TtL were expressed in Saccharomyces cerevisiae, and secreted enzymes were purified by consecutive hydrophobic and gel chromatography. The molecular masses of TtL (~ 65 kDa) and MtL (> 100 kDa) suggested glycosylation of the recombinant enzymes. Deglycosylation of MtL and TtL led to 25% and 10% decreases in activity, respectively. In a thermal stability assay, TtL retained 61% and MtL 86% of the initial activity at 40 °C. While TtL retained roughly 50% activity at 60 °C, MtL lost stability at temperatures higher than 40 °C. MtL and TtL preferred syringaldazine as a substrate, and the catalytic efficiencies for ABTS oxidation were 7.5 times lower than for syringaldazine oxidation. In the presence of the mediator HBT, purified TtL almost completely decolorized dyes within 30 min and substantially decolorized wastewater samples from a textile factory (up to 74%) within 20 h. However, products of TtL-catalyzed decolorization were more toxic than MtL-decolorized products, which were almost completely detoxified.

Crystal structure of carbonic anhydrase CaNce103p from the pathogenic yeast Candida albicans.

BACKGROUND: The pathogenic yeast Candida albicans can proliferate in environments with different carbon dioxide concentrations thanks to the carbonic anhydrase CaNce103p, which accelerates spontaneous conversion of carbon dioxide to bicarbonate and vice versa. Without functional CaNce103p, C. albicans cannot survive in atmospheric air. CaNce103p falls into the ß-carbonic anhydrase class, along with its ortholog ScNce103p from Saccharomyces cerevisiae. The crystal structure of CaNce103p is of interest because this enzyme is a potential target for surface disinfectants. RESULTS: Recombinant CaNce103p was prepared in E. coli, and its crystal structure was determined at 2.2 Å resolution. CaNce103p forms a homotetramer organized as a dimer of dimers, in which the dimerization and tetramerization surfaces are perpendicular. Although the physiological role of CaNce103p is similar to that of ScNce103p from baker's yeast, on the structural level it more closely resembles carbonic anhydrase from the saprophytic fungus Sordaria macrospora, which is also tetrameric. Dimerization is mediated by two helices in the N-terminal domain of the subunits. The N-terminus of CaNce103p is flexible, and crystals were obtained only upon truncation of the first 29 amino acids. Analysis of CaNce103p variants truncated by 29, 48 and 61 amino acids showed that residues 30-48 are essential for dimerization. Each subunit contains a zinc atom in the active site and displays features characteristic of type I ß-carbonic anhydrases. Zinc is tetrahedrally coordinated by one histidine residue, two cysteine residues and a molecule of ß-mercaptoethanol originating from the crystallization buffer. The active sites are accessible via substrate tunnels, which are slightly longer and narrower than those observed in other fungal carbonic anhydrases. CONCLUSIONS: CaNce103p is a ß-class homotetrameric metalloenzyme composed of two homodimers. Its structure closely resembles those of other ß-type carbonic anhydrases, in particular CAS1 from Sordaria macrospora. The main differences occur in the N-terminal part and the substrate tunnel. Detailed knowledge of the CaNce103p structure and the properties of the substrate tunnel in particular will facilitate design of selective inhibitors of this enzyme.

Vaginal use of micronized progesterone for luteal support.A randomized study comparing Utrogestan® and Crinone® 8.

BACKGROUND AND OBJECTIVE: Luteal phase physiology is distorted by in vitro fertilization (IVF) cycles using gonadotropin-releasing hormone (GnRH) agonists and antagonists, Controlled ovarian hyperstimulation leads to luteal phase defect and for this reason, luteal phase support is now an integral part of IVF/ICSI-ET programs. The support is provided by hCG, progesterone or GnRH-a. This study compared the efficiency, safety and tolerance of two vaginal micronized progesterones, Utrogestan and Crinone 8%. METHODS: 111 women, 18-40 years old, FSH < 10 IU/L and normal uterus findings were included. The efficiency of the two preparations to provide luteal support was evaluated by the fertilization, implantation, pregnancy and take-home baby rates. The safety was compared through the results of vaginal findings and vaginal inflammation markers before and after treatment. Comparison of tolerance was made by evaluating 21 subjective patient questionnaire parameters. RESULTS: There were no significant differences between the preparations in terms of efficiency or safety though Crinone 8% was better tolerated. CONCLUSION: The outcomes of this study suggest that a vaginal gel with micronized progesterone (Crinone 8%) is the optimal choice at this time for luteal support.

The Role of Cysteine Residues in Catalysis of Phosphoenolpyruvate Carboxykinase from Mycobacterium tuberculosis.

Mycobacterium tuberculosis (MTb), the causative agent of tuberculosis, can persist in macrophages for decades, maintaining its basic metabolic activities. Phosphoenolpyruvate carboxykinase (Pck; EC 4.1.1.32) is a key player in central carbon metabolism regulation. In replicating MTb, Pck is associated with gluconeogenesis, but in non-replicating MTb, it also catalyzes the reverse anaplerotic reaction. Here, we explored the role of selected cysteine residues in function of MTb Pck under different redox conditions. Using mass spectrometry analysis we confirmed formation of S-S bridge between cysteines C391 and C397 localized in the C-terminal subdomain. Molecular dynamics simulations of C391-C397 bridged model indicated local conformation changes needed for formation of the disulfide. Further, we used circular dichroism and Raman spectroscopy to analyze the influence of C391 and C397 mutations on Pck secondary and tertiary structures, and on enzyme activity and specificity. We demonstrate the regulatory role of C391 and C397 that form the S-S bridge and in the reduced form stabilize Pck tertiary structure and conformation for gluconeogenic and anaplerotic reactions.

Myristoylation drives dimerization of matrix protein from mouse mammary tumor virus.

BACKGROUND: Myristoylation of the matrix (MA) domain mediates the transport and binding of Gag polyproteins to the plasma membrane (PM) and is required for the assembly of most retroviruses. In betaretroviruses, which assemble immature particles in the cytoplasm, myristoylation is dispensable for assembly but is crucial for particle transport to the PM. Oligomerization of HIV-1 MA stimulates the transition of the myristoyl group from a sequestered to an exposed conformation, which is more accessible for membrane binding. However, for other retroviruses, the effect of MA oligomerization on myristoyl group exposure has not been thoroughly investigated. RESULTS: Here, we demonstrate that MA from the betaretrovirus mouse mammary tumor virus (MMTV) forms dimers in solution and that this process is stimulated by its myristoylation. The crystal structure of N-myristoylated MMTV MA, determined at 1.57 Å resolution, revealed that the myristoyl groups are buried in a hydrophobic pocket at the dimer interface and contribute to dimer formation. Interestingly, the myristoyl groups in the dimer are mutually swapped to achieve energetically stable binding, as documented by molecular dynamics modeling. Mutations within the myristoyl binding site resulted in reduced MA dimerization and extracellular particle release. CONCLUSIONS: Based on our experimental, structural, and computational data, we propose a model for dimerization of MMTV MA in which myristoyl groups stimulate the interaction between MA molecules. Moreover, dimer-forming MA molecules adopt a sequestered conformation with their myristoyl groups entirely buried within the interaction interface. Although this differs from the current model proposed for lentiviruses, in which oligomerization of MA triggers exposure of myristoyl group, it appears convenient for intracellular assembly, which involves no apparent membrane interaction and allows the myristoyl group to be sequestered during oligomerization.

Atomic resolution crystal structure of Sapp2p, a secreted aspartic protease from Candida parapsilosis.

The virulence of the Candida pathogens is enhanced by the production of secreted aspartic proteases, which therefore represent possible targets for drug design. Here, the crystal structure of the secreted aspartic protease Sapp2p from Candida parapsilosis was determined. Sapp2p was isolated from its natural source and crystallized in complex with pepstatin A, a classical aspartic protease inhibitor. The atomic resolution of 0.83 Šallowed the protonation states of the active-site residues to be inferred. A detailed comparison of the structure of Sapp2p with the structure of Sapp1p, the most abundant C. parapsilosis secreted aspartic protease, was performed. The analysis, which included advanced quantum-chemical interaction-energy calculations, uncovered molecular details that allowed the experimentally observed equipotent inhibition of both isoenzymes by pepstatin A to be rationalized.

Enzymological description of multitemplate PCR-Shrinking amplification bias by optimizing the polymerase-template ratio.

Multitemplate polymerase chain reaction (PCR) is used for preparative and analytical applications in diagnostics and research. Classical PCR and qPCR are two basic setups with many possible experimental modifications. Classical PCR is a method of choice to obtain enough material for subsequent sophisticated applications such as construction of libraries for next-generation sequencing or high-throughput screening. Sequencing and Single Nucleotide Primer Extension (SNuPE) employ one-strand synthesis and represent a distinct variant of analytical DNA synthesis. In all these applications, maintaining the initial ratio of templates and avoiding underestimation of minority templates is desired. Here, we demonstrate that different templates can amplify independently at low template concentrations (typical in qPCR setups, in which the polymerase concentration is usually several orders of magnitude higher than the template concentration). However, rare templates can be diluted in an effort to keep DNA amplification in the exponential phase, or template concentration can be biased by differences in amplification efficiency. Moreover, amplification of templates present in low concentrations is more vulnerable to stochastic events that lead to proportional changes in the product ratio, as well as by incomplete amplification leading to chimera formation. These undesired effects can be compensated for by using highly processive polymerases with high and equal affinity to different primer-template complexes. Novel enhanced polymerases are desired. With increasing concentration of a primer-template of interest, the system becomes more deterministic. Nevertheless, marked deviation from independent exponential amplification occurs when the total template concentration starts to approach the polymerase concentration. The primer-template complexes compete for enzyme molecules, and the amount of products grows arithmetically-the system starts to obey Michaelis-Menten kinetics. Synthesis of rare products in a multitemplate mixture can run more easily under the detection limit in such conditions, although it would be unequivocally detectable in a single template assay. When fishing out rare template variants, the best processive polymerases should be used to decrease both amplification and detection limits. The possibility of stochastic events, should be taken into account to correctly interpret the obtained data.