Mitogenomic phylogeny of the Percichthyidae and Centrarchiformes (Percomorphaceae): comparison with recent nuclear gene-based studies and simultaneous analysis

Delineation of the fish family Percichthyidae (Percomorphaceae) has a long and convoluted history, with recent morphological-based studies restricting species members to South American and Australian freshwater and catadromous temperate perches. Four recent nuclear gene-based phylogenetic studies, however, found that the Percichthyidae was not monophyletic and was nested within a newly discovered inter-familial clade of Percomorphaceae, the Centrarchiformes, which comprises the Centrarchidae and 12 other families. Here, we reexamined the systematics of the Percichthyidae and Centrarchiformes based on new mitogenomic information. Our mitogenomic results are globally congruent with the recent nuclear gene-based studies although the overall amount of phylogenetic signal of the mitogenome is lower. They do not support the monophyly of the Percichthyidae, because the catadromous genus Percalates is not exclusively related to the freshwater percichthyids. The Percichthyidae (minus Percalates) and Percalates belong to a larger clade, equivalent to the Centrarchiformes, but their respective sister groups are unresolved. Because all recent analyses recover a monophyletic Centrarchiformes but with substantially different intra-relationships, we performed a simultaneous analysis for a character set combining the mitogenome and 19 nuclear genes previously published, for 22 centrarchiform taxa. This analysis furthermore indicates that the Centrarchiformes are divided into three lineages and the superfamily Cirrhitoidea is monophyletic as well as the temperate and freshwater centrarchiform perch-like fishes. It also clarifies some of the relationships within the freshwater Percichthyidae.     

Oxygen-binding Heme Complexes of Peptides Designed to Mimic the Heme Environment of Myoglobin and Hemoglobin

Development of effective resuscitation agents for blood-loss replacement in trauma or surgery is extremely important despite substantial improvements in screening methods of blood from human donors. This paper reports the design and synthesis of peptides that mimic the natural environment of the heme group in myoglobin (Mb) and in the - and -subunits of human adult hemoglobin (Hb). The designs were based on the fact that the heme group in the aforementioned proteins is sandwiched between helices E and F. Fifteen test peptides and six control peptides were synthesized, and their ability to form stable complexes with heme was investigated. It was found that none of the control peptides or proteins was able to bind heme. However, each of the peptides that were designed to mimic the E--F helices, and even shorter designs, which removed from this region residues that do not contribute to contacts with the heme group, were each able to bind one mole of heme per mole of peptide forming peptide–heme complexes that were stable to manipulation and behaved as single molecular species. Oxygen binding measurements on the reduced peptide–heme complexes showed that these compounds bind oxygen and give visible spectra that were typical of oxygenated heme-proteins. In oxygen binding measurements done under different partial pressures of oxygen, the heme–peptide complexes gave hyperbolic oxygen-saturation curves, but showed slight differences in their P₅₀ values. The P₅₀ values ranged from 3.8 mmHg for the heme–peptide B7 complex to 13.7 mmHg for the heme–peptide D13 complex (under the same conditions, P₅₀ values for Hb and Mb were 34.0 and 5.5 mmHg, respectively). It is concluded that peptide constructs designed to mimic the heme-binding regions of Mb or the Hb subunits were able to form coordinate 1:1 complexes with heme, and these complexes bind oxygen in a manner expected for single subunit heme proteins.     

Diagnostic accuracy of fine-needle aspiration cytology in histological grade 1 breast carcinomas: are we good enough?

Background: Fine-needle aspiration cytology (FNAC) of both palpable and non-palpable breast carcinomas has a high accuracy and sensitivity in dedicated centres. It is generally thought that low-grade carcinomas have a distinctly lower sensitivity due to discrete cellular atypia that may be difficult to appreciate. Grade 1 carcinomas make up about 45% of screening-detected breast carcinomas and about 20% of symptomatic breast cancers. The aim of this study was to evaluate the diagnostic sensitivity of grade 1 carcinomas and identify the critical features in the cytological diagnostic work-up of these tumours. Methods: There were FNAC smears from 494 histologically confirmed grade 1 carcinomas diagnosed during 1996–2004. The cytological diagnoses were compared with the histology. Results: A definitive malignant diagnosis (absolute sensitivity) was given in 382 cases (77.3%). Equivocal or suspicious diagnoses were given in 75 (15.2%), benign or probably benign (false negative) in 24 (4.8%). Thirteen cases (2.6%) were unsatisfactory. Complete sensitivity was 92.7%. Invasive ductal carcinomas comprised 81.3% of all cases; absolute sensitivity for these was 80.9%. Invasive lobular and tubular carcinomas comprised 7.3% and 5.9% of cases, respectively; absolute sensitivity for these diagnosis was 50.0% and 57.1%, respectively, significantly lower than for other subtypes (P ≤ 0.0001) whereas the difference for complete sensitivity was less but still significant (P = 0.017). Absolute and complete sensitivities were lower for tumours less than 1 cm size compared with more than 1 cm (P ≤ 0.00001). Conclusion: Preoperative FNAC diagnosis of grade 1 breast carcinoma has a high sensitivity, especially in ductal carcinomas. Invasive lobular and tubular carcinomas were less likely to receive a definite preoperative diagnosis. The main reason for not reaching a definitive malignant diagnosis was sampling error due to small tumours less than 1 cm in diameter, irrespective of tumour subtype.     

Improving the aqueous solubility of HCV‐E2 glycoprotein epitope mimics by cyclization using POLAR hinges

In this research we describe the improvement of the water‐solubility of cyclic epitope mimics based on the HCV E2 glycoprotein by incorporation of suitable polar hinges. The poor solubility of epitope mimics based on peptide sequences in the envelope (E2) protein hampered their synthesis and purification and made it very difficult to prepare the molecular constructs for evaluation of their bioactivity. Since changes in the amino acid composition are hardly possible in these epitope mimics in order to increase water‐solubility, a polar cyclization hinge may offer a remedy leading to a significant increase of polarity and therefore water solubility. These polar hinges were applied in the synthesis of better water‐soluble HCV‐E2 epitopes. An azide functionality in the polar hinges allowed attachment of a tetraethylene glycol linker by Cu‐catalyzed azide‐alkyne cyclo‐addition (CuAAC) for a convenient conjugation to ELISA plates in order to evaluate the bio‐activity of the epitope mimics. The immunoassays showed that the use of more polar cyclization hinges still supported anti‐HCV antibody recognition and did not negatively influence their binding. This significantly increased solubility induced by polar hinges should therefore allow for the molecular construction and ultimate evaluation of synthetic vaccine molecules.     

A Site‐Specific Integration Reporter System That Enables Rapid Evaluation of CRISPR/Cas9‐Mediated Genome Editing Strategies in CHO Cells

Targeted gene knockout and site‐specific integration (SSI) are powerful genome editing techniques to improve the development of industrially relevant Chinese hamster ovary (CHO) cell lines. However, past efforts to perform SSI in CHO cells are characterized by low efficiencies. Moreover, numerous strategies proposed to boost SSI efficiency in mammalian cell types have yet to be evaluated head to head or in combination to appreciably boost efficiencies in CHO. To enable systematic and rapid optimization of genome editing methods, the SSIGNAL (s ite‐s pecific i ntegration and g en ome al teration) reporter system is developed. This tool can analyze CRISPR (clustered regularly interspaced palindromic repeats)/Cas9 (CRISPR‐associated protein 9)‐mediated disruption activity alone or in conjunction with SSI efficiency. The reporter system uses green and red dual‐fluorescence signals to indicate genotype states within four days following transfection, facilitating rapid data acquisition via standard flow cytometry instrumentation. In addition to describing the design and development of the system, two of its applications are demonstrated by first comparing transfection conditions to maximize CRISPR/Cas9 activity and subsequently assessing the efficiency of several promising SSI strategies. Due to its sensitivity and versatility, the SSIGNAL reporter system may serve as a tool to advance genome editing technology.     

The enigmatic Crimean green lizard (Lacerta viridis magnifica) is extinct but not valid: Mitogenomics of a 120-year-old museum specimen reveals historical introduction

It has been proposed that Lacerta viridis magnifica Sobolevssky, 1930 represents an extinct species or subspecies of green lizard endemic to the southern Crimea. Using NGS protocols optimized for heavily degraded DNA, we sequenced the complete mitogenome of one of the originally formalin-preserved specimens collected in the late 19th century. A comparison with sequence data of other green lizards revealed that L. v. magnifica is a junior synonym of the northern subspecies of the western green lizard (L. b. bilineata Daudin, 1802), which occurs at least 1,500 km away, beyond the distribution ranges of other green lizards. In medieval times, a Genoese colony existed in the Crimean region where the extinct green lizards occurred. Until the early 20th century, close ties to Italy persisted, and locals of Genoese descent sent their children for education to Italy, where L. b. bilineata occurs. This suggests that the extinct Crimean green lizards have been introduced accidentally or intentionally from Italy. Our study exemplifies the value of historical formalin-preserved museum specimens for clarifying the status of questionable rare or extinct taxa.     

Designer Condensates: A Toolkit for the Biomolecular Architect

Protein phase separation has emerged as a novel paradigm to explain the biogenesis of membraneless organelles and other so-called biomolecular condensates. While the implication of this physical phenomenon within cell biology is providing us with novel ways for understanding how cells compartmentalize biochemical reactions and encode function in such liquid-like assemblies, the newfound appreciation of this process also provides immense opportunities for designing and sculpting biological matter. Here, we propose that understanding the cell’s instruction manual of phase separation will enable bioengineers to begin creating novel functionalized biological materials and unprecedented tools for synthetic biology. We present FASE as the synthesis of the existing sticker-spacer framework, which explains the physical driving forces underlying phase separation, with quintessential principles of Scandinavian design. FASE serves both as a designer condensates catalogue and construction manual for the aspiring (membraneless) biomolecular architect. Our approach aims to inspire a new generation of bioengineers to rethink phase separation as an opportunity for creating reactive biomaterials with unconventional properties and to encode novel biological function in living systems. Although still in its infancy, several studies highlight how designer condensates have immediate and widespread potential applications in industry and medicine.