Three-column osteotomy in long constructs has lower rates of proximal junctional kyphosis and better restoration of lumbar lordosis than anterior column realignment.

PURPOSE: Three-column osteotomies (TCOs) and minimally invasive techniques such as anterior column realignment (ACR) are powerful tools used to restore lumbar lordosis and sagittal alignment. We aimed to appraise the differences in construct and global spinal stability between TCOs and ACRs in long constructs. METHODS: We identified consecutive patients who underwent a long construct lumbar or thoracolumbar fusion between January 2016 and November 2021. "Long construct" was any construct where the uppermost instrumented vertebra (UIV) was L2 or higher and the lowermost instrumented vertebra (LIV) was in the sacrum or ileum. RESULTS: We identified 69 patients; 14 (20.3%) developed PJK throughout follow-up (mean 838 days). Female patients were less likely to suffer PJK (p = 0.009). TCO was more associated with open (versus minimally invasive) screw/rod placement, greater number of levels, higher UIV, greater rate of instrumentation to the ilium, and posterior (versus anterior) L5-S1 interbody placement versus the ACR cohort (p < 0.001, p < 0.001, p < 0.001, p < 0.001, p = 0.005, respectively). Patients who developed PJK were more likely to have undergone ACR (12 (32.4%) versus 2 (6.3%, p = 0.007)). The TCO cohort had better improvement of lumbar lordosis despite similar preoperative measurements (ACR: 16.8 ± 3.78°, TCO: 23.0 ± 5.02°, p = 0.046). Pelvic incidence-lumbar lordosis mismatch had greater improvement after TCO (ACR: 14.8 ± 4.02°, TCO: 21.5 ± 5.10°, p = 0.042). By multivariate analysis, ACR increased odds of PJK by 6.1-times (95% confidence interval: 1.20-31.2, p = 0.29). CONCLUSION: In patients with long constructs who undergo ACR or TCO, we experienced a 20% rate of PJK. TCO decreased PJK 6.1-times compared to ACR. TCO demonstrated greater improvement of some spinopelvic parameters.

Phylogeographic inference of Sumatran ranids bearing gastromyzophorous tadpoles with regard to the Pleistocene drainage systems of Sundaland.

Rivers are known to act as biogeographic barriers in several strictly terrestrial taxa, while possibly serving as conduits of dispersal for freshwater-tolerant or -dependent species. However, the influence of river systems on genetic diversity depends on taxa-specific life history traits as well as other geographic factors. In amphibians, several studies have demonstrated that river systems have only minor influence on their divergence. Here, we assess the role of the paleodrainage systems of the Sunda region (with a focus on the island of Sumatra) in shaping the evolutionary history of two genera of frogs (Sumaterana and Wijayarana) whose tadpoles are highly dependent on cascading stream habitats. Our phylogenetic results show no clear association between the genetic diversification patterns of both anurans genera and the existence of paleodrainage systems. Time-calibrated phylogenies and biogeographical models suggest that these frogs colonized Sumatra and diversified on the island before the occurrence of the Pleistocene drainage systems. Both genera demonstrate phylogenetic structuring along a north-south geographic axis, the temporal dynamics of which coincide with the geological chronology of proto Sumatran and -Javan volcanic islands. Our results also highlight the chronic underestimation of Sumatran biodiversity and call for more intense sampling efforts on the island.

Unexpectedly high levels of lineage diversity in Sundaland puddle frogs (Dicroglossidae: Occidozyga Kuhl and van Hasselt, 1822).

One of the most urgent contemporary tasks for taxonomists and evolutionary biologists is to estimate the number of species on earth. Recording alpha diversity is crucial for protecting biodiversity, especially in areas of elevated species richness, which coincide geographically with increased anthropogenic environmental pressures - the world's so-called biodiversity hotspots. Although the distribution of Puddle frogs of the genus Occidozyga in South and Southeast Asia includes five biodiversity hotspots, the available data on phylogeny, species diversity, and biogeography are surprisingly patchy. Samples analyzed in this study were collected throughout Southeast Asia, with a primary focus on Sundaland and the Philippines. A mitochondrial gene region comprising ~ 2000 bp of 12S and 16S rRNA with intervening tRNA Valine and three nuclear loci (BDNF, NTF3, POMC) were analyzed to obtain a robust, time-calibrated phylogenetic hypothesis. We found a surprisingly high level of genetic diversity within Occidozyga, based on uncorrected p-distance values corroborated by species delimitation analyses. This extensive genetic diversity revealed 29 evolutionary lineages, defined by the > 5% uncorrected p-distance criterion for the 16S rRNA gene, suggesting that species diversity in this clade of phenotypically homogeneous forms probably has been underestimated. The comparison with results of other anuran groups leads to the assumption that anuran species diversity could still be substantially underestimated in Southeast Asia in general. Many genetically divergent lineages of frogs are phenotypically similar, indicating a tendency towards extensive morphological conservatism. We present a biogeographic reconstruction of the colonization of Sundaland and nearby islands which, together with our temporal framework, suggests that lineage diversification centered on the landmasses of the northern Sunda Shelf. This remarkably genetically structured group of amphibians could represent an exceptional case for future studies of geographical structure and diversification in a widespread anuran clade spanning some of the most pronounced geographical barriers on the planet (e.g., Wallace's Line). Studies considering gene flow, morphology, ecological and bioacoustic data are needed to answer these questions and to test whether observed diversity of Puddle frog lineages warrants taxonomic recognition.

Anatomical features of the phytotelma dwelling, egg-eating, fanged tadpoles of Rhacophorus vampyrus (Anura: Rhacophoridae).

Tadpoles of the Vampire tree frog Rhacophorus vampyrus differ substantially from other rhacophorid tadpoles, by having profound modifications in external morphology. The morphological peculiarities of this species likely correlate with their arboreal microhabitat and strict oophagous diet. In this work, we examine buccal and musculoskeletal anatomy and compare them to other rhacophorid and egg-eating larvae. The shape and arrangement of cartilages of the lower jaw are unique among tadpoles, and the lack of a palatoquadrate suspensorium is only known in the distantly related macrophagous tadpoles of the dicroglossid Occidozyga baluensis. The cranial musculature is massive, and the morphology of several mandibular, hyoid, and abdominal muscles could be related to the ingestion and transit of large eggs. In the buccal cavity, conspicuous aspects are the absence of ridges and papillae, and the development of a unique glandular zone in the buccal floor. Finally, observations of the skeletal support of keratinized mouthparts allow us to present a topography-based hypothesis of homology of the conspicuous fangs of these tadpoles.

Wnt10b-GSK3ß-dependent Wnt/STOP signaling prevents aneuploidy in human somatic cells.

Wnt signaling is crucial for proper development, tissue homeostasis and cell cycle regulation. A key role of Wnt signaling is the GSK3ß-mediated stabilization of ß-catenin, which mediates many of the critical roles of Wnt signaling. In addition, it was recently revealed that Wnt signaling can also act independently of ß-catenin. In fact, Wnt mediated stabilization of proteins (Wnt/STOP) that involves an LRP6-DVL-dependent signaling cascade is required for proper regulation of mitosis and for faithful chromosome segregation in human somatic cells. We show that inhibition of Wnt/LRP6 signaling causes whole chromosome missegregation and aneuploidy by triggering abnormally increased microtubule growth rates in mitotic spindles, and this is mediated by increased GSK3ß activity. We demonstrate that proper mitosis and maintenance of numerical chromosome stability requires continuous basal autocrine Wnt signaling that involves secretion of Wnts. Importantly, we identified Wnt10b as a Wnt ligand required for the maintenance of normal mitotic microtubule dynamics and for proper chromosome segregation. Thus, a self-maintaining Wnt10b-GSK3ß-driven cellular machinery ensures the proper execution of mitosis and karyotype stability in human somatic cells.

Ecomorphology of the pectoral girdle in anurans (Amphibia, Anura): Shape diversity and biomechanical considerations.

Frogs and toads (Lissamphibia: Anura) show a diversity of locomotor modes that allow them to inhabit a wide range of habitats. The different locomotor modes are likely to be linked to anatomical specializations of the skeleton within the typical frog Bauplan. While such anatomical adaptations of the hind limbs and the pelvic girdle are comparably well understood, the pectoral girdle received much less attention in the past. We tested for locomotor-mode-related shape differences in the pectoral girdle bones of 64 anuran species by means of micro-computed-tomography-based geometric morphometrics. The pectoral girdles of selected species were analyzed with regard to the effects of shape differences on muscle moment arms across the shoulder joint and stress dissipation within the coracoid. Phylogenetic relationships, size, and locomotor behavior have an effect on the shape of the pectoral girdle in anurans, but there are differences in the relative impact of these factors between the bones of this skeletal unit. Remarkable shape diversity has been observed within locomotor groups indicating many-to-one mapping of form onto function. Significant shape differences have mainly been related to the overall pectoral girdle geometry and the shape of the coracoid. Most prominent shape differences have been found between burrowing and nonburrowing species with headfirst and backward burrowing species significantly differing from one another and from the other locomotor groups. The pectoral girdle shapes of burrowing species have generally larger moment arms for (simulated) humerus retractor muscles across the shoulder joint, which might be an adaptation to the burrowing behavior. The mechanisms of how the moment arms were enlarged differed between species and were associated with differences in the reaction of the coracoid to simulated loading by physiologically relevant forces.

A little frog leaps a long way: compounded colonizations of the Indian Subcontinent discovered in the tiny Oriental frog genus Microhyla (Amphibia: Microhylidae).

Frogs of the genus Microhyla include some of the world's smallest amphibians and represent the largest radiation of Asian microhylids, currently encompassing 50 species, distributed across the Oriental biogeographic region. The genus Microhyla remains one of the taxonomically most challenging groups of Asian frogs and was found to be paraphyletic with respect to large-sized fossorial Glyphoglossus. In this study we present a time-calibrated phylogeny for frogs in the genus Microhyla, and discuss taxonomy, historical biogeography, and morphological evolution of these frogs. Our updated phylogeny of the genus with nearly complete taxon sampling includes 48 nominal Microhyla species and several undescribed candidate species. Phylogenetic analyses of 3,207 bp of combined mtDNA and nuDNA data recovered three well-supported groups: the Glyphoglossus clade, Southeast Asian Microhyla II clade (includes M. annectens species group), and a diverse Microhyla I clade including all other species. Within the largest major clade of Microhyla are seven well-supported subclades that we identify as the M. achatina, M. fissipes, M. berdmorei, M. superciliaris, M. ornata, M. butleri, and M. palmipes species groups. The phylogenetic position of 12 poorly known Microhyla species is clarified for the first time. These phylogenetic results, along with molecular clock and ancestral area analyses, show the Microhyla-Glyphoglossus assemblage to have originated in Southeast Asia in the middle Eocene just after the first hypothesized land connections between the Indian Plate and the Asian mainland. While Glyphoglossus and Microhyla II remained within their ancestral ranges, Microhyla I expanded its distribution generally east to west, colonizing and diversifying through the Cenozoic. The Indian Subcontinent was colonized by members of five Microhyla species groups independently, starting with the end Oligocene-early Miocene that coincides with an onset of seasonally dry climates in South Asia. Body size evolution modeling suggests that four groups of Microhyla have independently achieved extreme miniaturization with adult body size below 15 mm. Three of the five smallest Microhyla species are obligate phytotelm-breeders and we argue that their peculiar reproductive biology may be a factor involved in miniaturization. Body size increases in Microhyla-Glyphoglossus seem to be associated with a burrowing adaptation to seasonally dry habitats. Species delimitation analyses suggest a vast underestimation of species richness and diversity in Microhyla and reveal 15-33 undescribed species. We revalidate M. nepenthicola, synonymize M. pulverata with M. marmorata, and provide insights on taxonomic statuses of a number of poorly known species. Further integrative studies, combining evidence from phylogeny, morphology, advertisement calls, and behavior will result in a better systematic understanding of this morphologically cryptic radiation of Asian frogs.

Measurement error in µCT-based three-dimensional geometric morphometrics introduced by surface generation and landmark data acquisition.

Computed-tomography-derived (CT-derived) polymesh surfaces are widely used in geometric morphometric studies. This approach is inevitably associated with decisions on scanning parameters, resolution, and segmentation strategies. Although the underlying processing steps have been shown to potentially contribute artefactual variance to three-dimensional landmark coordinates, their effects on measurement error have rarely been assessed systematically in CT-based geometric morphometric studies. The present study systematically assessed artefactual variance in landmark data introduced by the use of different voxel sizes, segmentation strategies, surface simplification degrees, and by inter- and intra-observer differences, and compared their magnitude to true biological variation. Multiple CT-derived surface variants of the anuran (Amphibia: Anura) pectoral girdle were generated by systematic changes in the factors that potentially influence the surface geometries. Twenty-four landmarks were repeatedly acquired by different observers. The contribution of all factors to the total variance in the landmark data was assessed using random-factor nested permanovas. Selected sets of Euclidean distances between landmark sets served further to compare the variance among factor levels. Landmark precision was assessed by landmark standard deviation and compared among observers and days. Results showed that all factors, except for voxel size, significantly contributed to measurement error in at least some of the analyses performed. In total, 6.75% of the variance in landmark data that mimicked a realistic biological study was caused by measurement error. In this landmark dataset, intra-observer error was the major source of artefactual variance followed by inter-observer error; the factor segmentation contributed < 1% and slight surface simplification had no significant effect. Inter-observer error clearly exceeded intra-observer error in a different landmark dataset acquired by six partly inexperienced observers. The results suggest that intra-observer error can potentially be reduced by including a training period prior to the actual landmark acquisition task and by acquiring landmarks in as few sessions as possible. Additionally, the application of moderate and careful surface simplification and, potentially, also the use of case-specific optimal combinations of automatic local thresholding algorithms and parameters for segmentation can help reduce intra-observer error. If landmark data are to be acquired by several observers, it is important to ensure that all observers are consistent in landmark identification. Despite the significant amount of artefactual variance, we have shown that landmark data acquired from microCT-derived surfaces are precise enough to study the shape of anuran pectoral girdles. Yet, a systematic assessment of measurement error is advisable for all geometric morphometric studies.

Disruption of diphthamide synthesis genes and resulting toxin resistance as a robust technology for quantifying and optimizing CRISPR/Cas9-mediated gene editing.

We have devised an effective and robust method for the characterization of gene-editing events. The efficacy of editing-mediated mono- and bi-allelic gene inactivation and integration events is quantified based on colony counts. The combination of diphtheria toxin (DT) and puromycin (PM) selection enables analyses of 10,000-100,000 individual cells, assessing hundreds of clones with inactivated genes per experiment. Mono- and bi-allelic gene inactivation is differentiated by DT resistance, which occurs only upon bi-allelic inactivation. PM resistance indicates integration. The robustness and generalizability of the method were demonstrated by quantifying the frequency of gene inactivation and cassette integration under different editing approaches: CRISPR/Cas9-mediated complete inactivation was ~30-50-fold more frequent than cassette integration. Mono-allelic inactivation without integration occurred >100-fold more frequently than integration. Assessment of gRNA length confirmed 20mers to be most effective length for inactivation, while 16-18mers provided the highest overall integration efficacy. The overall efficacy was ~2-fold higher for CRISPR/Cas9 than for zinc-finger nuclease and was significantly increased upon modulation of non-homologous end joining or homology-directed repair. The frequencies and ratios of editing events were similar for two different DPH genes (independent of the target sequence or chromosomal location), which indicates that the optimization parameters identified with this method can be generalized.

A Comparison of the Anti-Inflammatory Effects of Cis-9, Trans-11 Conjugated Linoleic Acid to Celecoxib in the Collagen-Induced Arthritis Model.

Cyclooxygenase (COX)-2 inhibitors, such as celecoxib, for chronic inflammatory disease are associated with adverse health events, while cis-9, trans-11 (c9t11) conjugated linoleic acid (CLA) is anti-inflammatory without adverse events attributed to pure intake. Mechanistically, celecoxib and c9t11 disrupt the arachidonic acid cascade; however, the equivalency of anti-inflammatory effects between these compounds is unknown. Therefore, to test the hypothesis that 0.5% dietary c9t11 reduces inflammation equivalently to a celecoxib dose intended to treat rheumatoid arthritis (RA; 5 mg/kg bw), arthritic mice received diets containing one of the following supplements: 1% corn oil (CO, w/w), 0.5% c9t11 (>91% purity) +0.5% CO, or 1% CO + 0.5, 5, or 50 mg/kg bw celecoxib, and were assessed for changes in arthritic severity over 6 weeks. Overall, arthritic severity in mice fed c9t11 was reduced (34%, P < 0.01) while celecoxib doses (0.5, 5, 50 mg/kg) reduced arthritic severity (16, 56, 48%, respectively) compared to CO-fed arthritic mice. Linear regression of the celecoxib dose-response showed 0.5% c9t11 (570 mg/kg bw) reduced arthritic severity equivalently to 1.5 mg/kg celecoxib. Interleukin-6 (IL-6) was increased in paws of arthritic mice fed CO compared to shams, but was decreased in arthritic groups fed 0.5% c9t11 and 5 mg/kg celecoxib, compared to arthritic mice fed CO (Ps ≤ 0.05). Additionally, paw and plasma IL-10 levels in arthritic mice were decreased by 5 mg/kg celecoxib, but were unaffected by c9t11 compared to CO. Results suggest dietary c9t11 may be an effective adjunct to COX-2 inhibition for treating chronic inflammation.

Rapid Activation of Bone Morphogenic Protein 9 by Receptor-mediated Displacement of Pro-domains.

By non-covalent association after proteolytic cleavage, the pro-domains modulate the activities of the mature growth factor domains across the transforming growth factor-ß family. In the case of bone morphogenic protein 9 (BMP9), however, the pro-domains do not inhibit the bioactivity of the growth factor, and the BMP9·pro-domain complexes have equivalent biological activities as the BMP9 mature ligand dimers. By using real-time surface plasmon resonance, we could demonstrate that either binding of pro-domain-complexed BMP9 to type I receptor activin receptor-like kinase 1 (ALK1), type II receptors, co-receptor endoglin, or to mature BMP9 domain targeting antibodies leads to immediate and complete displacement of the pro-domains from the complex. Vice versa, pro-domain binding by an anti-pro-domain antibody results in release of the mature BMP9 growth factor. Based on these findings, we adjusted ELISA assays to measure the protein levels of different BMP9 variants. Although mature BMP9 and inactive precursor BMP9 protein were directly detectable by ELISA, BMP9·pro-domain complex could only be measured indirectly as dissociated fragments due to displacement of mature growth factor and pro-domains after antibody binding. Our studies provide a model in which BMP9 can be readily activated upon getting into contact with its receptors. This increases the understanding of the underlying biology of BMP9 activation and also provides guidance for ELISA development for the detection of circulating BMP9 variants.

Loss of diphthamide pre-activates NF-κB and death receptor pathways and renders MCF7 cells hypersensitive to tumor necrosis factor.

The diphthamide on human eukaryotic translation elongation factor 2 (eEF2) is the target of ADP ribosylating diphtheria toxin (DT) and Pseudomonas exotoxin A (PE). This modification is synthesized by seven dipthamide biosynthesis proteins (DPH1-DPH7) and is conserved among eukaryotes and archaea. We generated MCF7 breast cancer cell line-derived DPH gene knockout (ko) cells to assess the impact of complete or partial inactivation on diphthamide synthesis and toxin sensitivity, and to address the biological consequence of diphthamide deficiency. Cells with heterozygous gene inactivation still contained predominantly diphthamide-modified eEF2 and were as sensitive to PE and DT as parent cells. Thus, DPH gene copy number reduction does not affect overall diphthamide synthesis and toxin sensitivity. Complete inactivation of DPH1, DPH2, DPH4, and DPH5 generated viable cells without diphthamide. DPH1ko, DPH2ko, and DPH4ko harbored unmodified eEF2 and DPH5ko ACP- (diphthine-precursor) modified eEF2. Loss of diphthamide prevented ADP ribosylation of eEF2, rendered cells resistant to PE and DT, but does not affect sensitivity toward other protein synthesis inhibitors, such as saporin or cycloheximide. Surprisingly, cells without diphthamide (independent of which the DPH gene compromised) were presensitized toward nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) and death-receptor pathways without crossing lethal thresholds. In consequence, loss of diphthamide rendered cells hypersensitive toward TNF-mediated apoptosis. This finding suggests a role of diphthamide in modulating NF-κB, death receptor, or apoptosis pathways.

Description of the tadpoles of three rare species of megophryid frogs (Amphibia: Anura: Megophryidae) from Gunung Mulu, Sarawak, Malaysia.

The megophryid frogs Leptobrachella brevicrus, Leptolalax dringi and Megophrys dringi are species exclusively known  from highly localised areas in isolated mountain ranges on Borneo. The tadpoles and adults in this study were collected at the shared type locality for the three species in Gunung Mulu National Park, Sarawak, Malaysia (Borneo). The species identities of larvae were determined via comparison to syntopic adults using DNA barcoding techniques based on partial 16S rRNA mitochondrial gene sequences. The genetic data supported the status of the three taxa as valid species. Descriptions of colouration in life and after preservation, external morphological features, morphometric measurements and ecological notes in comparison to congeneric species are supplied. The tadpoles of L. brevicrus and L. dringi show similar adaptations to a fossorial lifestyle. These include an elongated, vermiform body, a relatively long tail and small eyes. Both were found in the gravel beds of a small mountain stream. In contrast, the larvae of M. dringi are adapted to occupying and feeding at the surface of pools within the stream. 

Dressed in black. A New Ansonia Stoliczka, 1870 (Lissamphibia: Anura: Bufonidae) from Gunung Murud, Sarawak, East Malaysia (Borneo).

A new species of stream toad of the genus Ansonia is described from Gunung Murud, Pulong Tau National Park, of northern Sarawak, Malaysia, Borneo. Ansonia vidua, sp. nov., is morphologically distinguished from its Bornean congeners by the following combination of characters: medium size (SVL of adult females 33.5-34.4 mm); body uniformly black-brown in life; absence of a visible pattern on dorsum or limbs; presence of two low interorbital ridges; shagreened skin on dorsum, sides and upper surfaces of the limbs with numerous homogeneously small, rounded warts; first finger shorter than second; reduced webbing between the toes and an absence of a sharp tarsal ridge. Uncorrected genetic distances between related taxa of > 4.3% in 16S rRNA gene support its status as a hitherto undescribed species.

Red hot chili pepper. A new Calluella stoliczka, 1872 (Lissamphibia: Anura: Microhylidae) from Sarawak, East Malaysia (Borneo).

A new brightly-coloured (olive and red) species of microhylid frog of the genus Calluella Stoliczka 1872 is described from the upper elevations of Gunung Penrissen and the Matang Range, Sarawak, East Malaysia (Borneo). Calluella capsa, new species, is diagnosable in showing the following combination of characters: SVL up to 36.0 mm; dorsum weakly granular; a faint dermal fold across forehead; toe tips obtuse; webbing on toes basal; lateral fringes on toes present; outer metatarsal tubercle present; and dorsum greyish-olive, with red spots; half of venter bright red, the rest with large white and dark areas. The new species is the eighth species of Calluella to be described, and the fourth known from Borneo. A preliminary phylogeny of Calluella and its relatives is presented, and the new taxon compared with congeners from Malaysia and other parts of south-east Asia. 

Anatomy, function, and evolution of jaw and hyobranchial muscles in cryptobranchoid salamander larvae.

Larval salamanders (Lissamphibia: Caudata) are known to be effective suction feeders in their aquatic environments, although they will eventually transform into terrestrial tongue feeding adults during metamorphosis. Early tetrapods may have had a similar biphasic life cycle and this makes larval salamanders a particularly interesting model to study the anatomy, function, development, and evolution of the feeding apparatus in terrestrial vertebrates. Here, we provide a description of the muscles that are involved in the feeding strike in salamander larvae of the Hynobiidae and compare them to larvae of the paedomorphic Cryptobranchidae. We provide a functional and evolutionary interpretation for the observed muscle characters. The cranial muscles in larvae from species of the Hynobiidae and Cryptobranchidae are generally very similar. Most notable are the differences in the presence of the m. hyomandibularis, a muscle that connects the hyobranchial apparatus with the lower jaw. We found this muscle only in Onychodactylus japonicus (Hynobiidae) but not in other hynobiid or cryptobranchid salamanders. Interestingly, the m. hyomandibularis in O. japonicus originates from the ceratobranchial I and not the ceratohyal, and thus exhibits what was previously assumed to be the derived condition. Finally, we applied a biomechanical model to simulate suction feeding in larval salamanders. We provide evidence that a flattened shape of the hyobranchial apparatus in its resting position is beneficial for a fast and successful suction feeding strike.

Diversification in a biodiversity hotspot--the evolution of Southeast Asian rhacophorid tree frogs on Borneo (Amphibia: Anura: Rhacophoridae).

The tree-frog family Rhacophoridae is a major group contributing to the high pecies richness and reproductive diversity among vertebrates of Sundaland. Nonetheless, rhacophorid evolution, specially on Borneo, has not been studied within a phylogenetic context. In this study, we examine the phylogenetic relationships of 38 (out of 41) Bornean species of Rhacophoridae, in combination with data from previous phylogenetic studies. In the final super matrix of 91 species, we analyse sequence data from two mitochondrial and three nuclear genes. The resulting trees show the genus Rhacophorus as a paraphyletic assemblage. As a consequence, we transfer Rhacophorus appendiculatus and R. kajau to two other genera and propose the new phylogeny-based combinations--Kurixalus appendiculatus and Feihyla kajau, respectively. Furthermore, we use our phylogenetic hypotheses to reconstruct the evolution of reproductive modes in rhacophorid tree frogs. Direct development to the exclusion of a free larval stage evolved twice independently, once in an ancestor of the Pseudophilautus+Raorchestes clade in India and Sri Lanka, and once within Philautus in Southeast Asia. The deposition of egg clutches covered by a layer of jelly in Feihyla is also present in F. kajau and thus confirms our generic reassignment. The remarkably high diversity of rhacophorid tree frogs on Borneo is the outcome of a complex pattern of repeated vicariance and dispersal events caused by past changes in the climatic and geological history of the Sunda shelf. We identified geographic clades of closely related endemic species within Rhacophorus and Philautus, which result from local island radiations on Borneo.

Hydrogen-bonded networks along and bifurcation of the E-pathway in quinol:fumarate reductase.

The E-pathway of transmembrane proton transfer has been demonstrated previously to be essential for catalysis by the diheme-containing quinol:fumarate reductase (QFR) of Wolinella succinogenes. Two constituents of this pathway, Glu-C180 and heme b(D) ring C (b(D)-C-) propionate, have been validated experimentally. Here, we identify further constituents of the E-pathway by analysis of molecular dynamics simulations. The redox state of heme groups has a crucial effect on the connectivity patterns of mobile internal water molecules that can transiently support proton transfer from the b(D)-C-propionate to Glu-C180. The short H-bonding paths formed in the reduced states can lead to high proton conduction rates and thus provide a plausible explanation for the required opening of the E-pathway in reduced QFR. We found evidence that the b(D)-C-propionate group is the previously postulated branching point connecting proton transfer to the E-pathway from the quinol-oxidation site via interactions with the heme b(D) ligand His-C44. An essential functional role of His-C44 is supported experimentally by site-directed mutagenesis resulting in its replacement with Glu. Although the H44E variant enzyme retains both heme groups, it is unable to catalyze quinol oxidation. All results obtained are relevant to the QFR enzymes from the human pathogens Campylobacter jejuni and Helicobacter pylori.

Bispecific antibody derivatives with restricted binding functionalities that are activated by proteolytic processing.

We have designed bispecific antibodies that bind one target (anti-Her3) in a bivalent IgG-like manner and contain one additional binding entity (anti-cMet) composed of one V(H) and one V(L) domain connected by a disulfide bond. The molecules are assembled by fusing a V(H,Cys44) domain via flexible connector peptides to the C-terminus of one H-chain (heavy chain), and a V(L,Cys100) to another H-chain. To ensure heterodimerization during expression in mammalian cells, we introduced complementary knobs-into-holes mutations into the different H-chains. The IgG-shaped trivalent molecules carry as third binding entity one disulfide-stabilized Fv (dsFv) without a linker between V(H) and V(L). Tethering the V(H) and V(L) domains at the C-terminus of the C(H)3 domain decreases the on-rates of the dsFv to target antigens without affecting off-rates. Steric hindrance resolves upon removal of one side of the double connection by proteolysis: this improves flexibility and accessibility of the dsFv and fully restores antigen access and affinity. This technology has multiple applications: (i) in cases where single-chain linkers are not desired, dsFvs without linkers can be generated by addition of furin site(s) in the connector that are processed during expression within mammalian cells; (ii) highly active (toxic) entities which affect expression can be produced as inactive dsFvs and subsequently be activated (e.g. via PreScission cleavage) during purification; (iii) entities can be generated which are targeted by the unrestricted binding entity and can be activated by proteases in target tissues. For example, Her3-binding molecules containing linkers with recognition sequences for matrix metalloproteases or urokinase, whose inactivated cMet binding site is activated by proteolytic processing.

Bispecific antibody derivatives based on full-length IgG formats.

Monoclonal antibodies have emerged as an effective therapeutic modality, and numerous antibodies have been approved for the treatment of several severe diseases or are currently in clinical development. To improve their therapeutic potential, monoclonal antibodies are constantly evolved by protein engineering. Particularly, the generation of bispecific antibodies raised special interest because of their ability to bind two different antigens at the same time, and the efficiency of these formats has been demonstrated in several clinical and preclinical studies. Up to now, the major drawbacks in using bispecific antibodies as a therapeutic agent have been difficult design and low-yield expression of homogeneous antibody populations. However, major technological improvements were made in protein engineering during the last years. This allows the design of several new IgG-based bispecific antibody formats that can be prepared in high yields and high homogeneity using conventional expression and purification techniques. Especially, recent development of IgG-fusions with disulfide-stabilized Fv fragments and of CrossMab-technologies facilitates the generation of bispecific antibodies with IgG-like architectures. Here we describe design principles and methods to express and purify different bispecific antibody formats derived from full-length IgGs.