hox gene expression predicts tetrapod-like axial regionalization in the skate,Leucoraja erinacea

The axial skeleton of tetrapods is organized into distinct anteroposterior regions of the vertebral column (cervical, trunk, sacral, and caudal), and transitions between these regions are determined by colinear anterior expression boundaries of Hox5/6, -9, -10, and -11 paralogy group genes within embryonic paraxial mesoderm. Fishes, conversely, exhibit little in the way of discrete axial regionalization, and this has led to scenarios of an origin of Hox-mediated axial skeletal complexity with the evolutionary transition to land in tetrapods. Here, combining geometric morphometric analysis of vertebral column morphology with cell lineage tracing of hox gene expression boundaries in developing embryos, we recover evidence of at least five distinct regions in the vertebral skeleton of a cartilaginous fish, the little skate (Leucoraja erinacea). We find that skate embryos exhibit tetrapod-like anteroposterior nesting of hox gene expression in their paraxial mesoderm, and we show that anterior expression boundaries of hox5/6, hox9, hox10, and hox11 paralogy group genes predict regional transitions in the differentiated skate axial skeleton. Our findings suggest that hox-based axial skeletal regionalization did not originate with tetrapods but rather has a much deeper evolutionary history than was previously appreciated.

The axial skeleton (vertebrae and ribs) is a defining feature of the vertebrate body form, and the existence of distinct axial skeletal regions along the anteroposterior (AP) axis of the body is thought to have facilitated evolutionary radiations and ecological specializations across vertebrate phylogeny (1–3). Tetrapod vertebral columns include at least four axial regions: cervical, trunk (further divided into thoracic and lumbar in most mammals and some reptiles), sacral, and caudal, the lengths of which have been modified in different tetrapod groups to suit various ecologies and lifestyles. In mammals and birds, vertebral regions are patterned by Hox gene expression within the paraxial mesoderm, and experimental manipulations to anterior Hox expression boundaries result in corresponding shifts in vertebral regional boundaries (4–8). For example, in mouse, the anterior expression limit of Hox10 genes aligns with the thoracic–lumbar transition, and in Hox10 paralogy group mutants the lumbar vertebrae undergo homeotic transformations to develop ribs, and therefore take on a thoracic identity (5). Additional comparative gene expression studies indicate that this role for Hox genes in patterning axial skeletal regions is likely conserved across tetrapods (9–14).In contrast, the vertebral skeletons of fishes are thought to be less regionalized than those of tetrapods. Historically, the axial skeletons of both bony and cartilaginous fishes were simply subdivided into trunk and tail regions (15–17), but ostariophysan teleosts also possess a Weberian apparatus, a set of specialized anterior vertebrae that transmit sound from the swim bladder to the inner ear (18). Variation in vertebral morphology along the axis, resulting in up to five regions, has also been documented in several actinopterygian species, including chinook salmon (Oncorhynchus tshawytscha) (19), ponyfish (Leiognathus equulus) (20), and the fossil Tarrasius problematicus (21), suggesting that axial regionalization might be more widespread among actinopterygians than previously thought.hox expression in the paraxial mesoderm has been examined in two teleost fishes with similar vertebral formulae—zebrafish (Danio rerio) and stickleback (Gasterosteus aculeatus)—and both taxa show nested expression along the AP axis (22, 23). In zebrafish, hox expression is initially dynamic but stabilizes at the 20-somite stage (22, 24). In both zebrafish and stickleback, the anterior limit of hoxc6 expression appears to mark the transition to the first rib-bearing vertebra, and in zebrafish the transition from rib-bearing to hemal arch-bearing (i.e., trunk to tail) vertebrae correlates with the anterior expression of hoxa12 (22, 25). Anterior expression limits of other hox genes from paralogy groups 7 to 11 all align with somites that contribute to the rib-bearing trunk region, however, and have no discrete anatomical correlates (22, 25–27). hox13 paralogs are expressed in the tail bud in zebrafish (28) and mutation of Hoxb13 results in the addition of two caudal vertebrae in mice (29), but there is no evidence relating hox13 expression to morphological changes in the caudal fin vertebrae of teleosts. These studies indicate that, despite evidence of some degree of axial regionalization in actinopterygians, teleost hox gene expression patterns are complex and difficult to relate to changes in vertebral morphology. Within cartilaginous fishes, nested AP hox expression has been demonstrated in the catshark (Scyliorhinus canicula) (30, 31). Moreover, regionalization of the axial skeleton of the thorny skate (Amblyraja radiata) was reported based on discrete morphological characteristics of vertebrae along the AP axis (32). However, evidence linking hox expression boundaries to changes in vertebral morphology along the axis in cartilaginous fishes is lacking. It therefore remains unclear what role Hox genes might have played in the evolution of vertebral complexity.To test whether the tetrapod Hox code—and its role in establishing axial regions within the vertebral skeleton—has a deeper evolutionary origin within vertebrates, we investigated hox gene expression and axial skeletal regionalization in a cartilaginous fish, the little skate (Leucoraja erinacea). As a sister taxon to bony vertebrates, comparative studies of skate development facilitate inference of anatomical and developmental conditions in the last common ancestor of jawed vertebrates, and our previous work on skate axial skeletal development allows us to confidently relate somite to vertebral identity along the AP axis (33–35). Contrary to previous assumptions of relative axial skeletal homogeneity, our quantitative assessment of morphology reveals that the skate vertebral column possesses at least five distinct axial regions, with regional boundaries predicted by tetrapod-like patterns of hox gene expression within embryonic somitic mesoderm. This suggests that core elements of the mechanism governing tetrapod axial skeletal regionalization have been conserved for ∼500 million y from the origin of jawed vertebrates.     

Cytotoxicity and bioavailability studies on a decoction of Oldenlandia diffusa and its fractions separated by HPLC

Aim of the study

Oldenlandia diffusa is a traditional Chinese herbal remedy with known cytotoxic activity in vitro and in vivo. The aim of the study was to identify the most cytotoxic constituents of a water extract (a decoction is traditionally used as a treatment) by HPLC and activity-guided fractionation. The bioavailability of the decoction and certain fractions, and the mode of cell death induced by these mixtures, were also investigated.

Materials and methods

A decoction of O. diffusa was prepared and separated by HPLC into eleven fractions (F1-11) for testing on the growth of HL60 leukaemia cells; two of the most active fractions were also tested on Caco-2 colon cancer cells. Cell viability was measured by trypan blue exclusion, DNA content (Cyquant NF assay) and neutral red uptake. Evidence of apoptosis was gained from cells stained with the nuclear dye DAPI, and detection of cleaved poly (ADP-ribose) polymerase (PARP) by Western blot.

Results

Fraction 9 was found to be the most active fraction, and, along with the decoction, induced apoptosis. Cells stained with DAPI showed a decrease in cell size and nuclear fragmentation characteristic of apoptosis. Detection of cleaved PARP further confirmed induction of apoptosis by O. diffusa decoction and fraction 9. The bioavailability of O. diffusa was investigated by production of post-absorption samples using Caco-2 intestinal epithelial monolayers. Addition of post-absorption samples (taken from the basolateral side after 3 h incubation with the decoction on the apical side) inhibited the growth of HL60 cells, and suggested a degree of bioavailability. The constituents in fraction 9 were further separated by HPLC and eight major compounds were identified by LC-MS: two of these were ursolic acid (UA) and its enantiomer oleanolic acid (OA). Concentrations of UA and OA in the decoction were then calculated by reference to the area of the peaks of UA and OA found in F9. The post-absorption sample of F9 contained six of the eight constituents in the original pre-absorption fraction 9.

Conclusions

Taken together, the results suggest that certain constituents, possibly including ursolic/oleanolic acid, may be bioavailable and at sufficient concentration to induce apoptosis in cancer cells in vitro through a mechanism including the cleavage of PARP.     

Mutations in purine nucleoside phosphorylase deficiency

Purine nucleoside phosphorylase deficiency is an inherited disease of purine metabolism characterized clinically as combined immunodeficiency. The molecular defects have been published for 4 different alleles in 3 patients. We report four new mutations including two amino acid substitutions, A 174P and G190V, a single codon deletion, ΔI129, and a point mutation in intron 3 which leads to aberrant splicing and creation of a premature stop codon in exon 4 (286 -18G→A). Of the previously reported mutations, E89K was found in one additional patient, and R234P was found in 3 unrelated patients, making R234P the most common mutation reported to date in this disease. Hum Mutat 9:118–121, 1997. © 1997 Wiley-Liss, Inc.     

Influence of Poor Oral Health on Physical Frailty: A Population‐Based Cohort Study of Older British Men

Objectives

To investigate the associations between objective and subjective measures of oral health and incident physical frailty.

Design

Cross‐sectional and longitudinal study with 3 years of follow‐up using data from the British Regional Heart Study.

Setting

General practices in 24 British towns.

Participants

Community‐dwelling men aged 71 to 92 (N = 1,622).

Measurements

Objective assessments of oral health included tooth count and periodontal disease. Self‐reported oral health measures included overall self‐rated oral health; dry mouth symptoms; sensitivity to hot, cold, and sweet; and perceived difficulty eating. Frailty was defined using the Fried phenotype as having 3 or more of weight loss, grip strength, exhaustion, slow walking speed, and low physical activity. Incident frailty was assessed after 3 years of follow‐up in 2014.

Results

Three hundred three (19%) men were frail at baseline (aged 71–92). Having fewer than 21 teeth, complete tooth loss, fair to poor self‐rated oral health, difficulty eating, dry mouth, and more oral health problems were associated with greater likelihood of being frail. Of 1,284 men followed for 3 years, 107 (10%) became frail. The risk of incident frailty was higher in participants who were edentulous (odds ratio (OR) = 1.90, 95% confidence interval (CI) = 1.03–3.52); had 3 or more dry mouth symptoms (OR = 2.03, 95% CI = 1.18–3.48); and had 1 (OR = 2.34, 95% CI = 1.18–4.64), 2 (OR = 2.30, 95% CI = 1.09–4.84), or 3 or more (OR = 2.72, 95% CI = 1.11–6.64) oral health problems after adjustment for age, smoking, social class, history of cardiovascular disease or diabetes mellitus, and medications related to dry mouth.

Conclusion

The presence of oral health problems was associated with greater risks of being frail and developing frailty in older age. The identification and management of poor oral health in older people could be important in preventing frailty.     

DA-EPOCH-R in Aggressive CD 20 Positive B Cell Lymphomas: Real-World Experience

Recent reports have shown that excellent survival outcomes can be achieved in adult Burkitt’s lymphoma with the use of DA-EPOCH-R regimen. When compared to earlier intense pediatric-type protocols, this regimen is less toxic. There are limited reports available on the use of this regimen outside the context of clinical trials. We analyzed the outcomes of patients who were treated with the DA-EPOCH-R regimen [Burkitt’s lymphoma (BL), primary mediastinal B cell lymphoma (PMBCL) and HIV-positive patients with diffuse large B cell lymphoma (DLBCL)] at our center over a 3 year period. Baseline characters, responses, and toxicity data was captured from records. Event-free survival (EFS—from therapy initiation till occurrence of event (non-achievement of complete response or relapse) and overall survival (OS—from therapy initiation till death due to any cause) were estimated using Kaplan–Meier method. Among 41 patients [median age 40 years (18–76)], the following diagnoses were included-HIV negative patients (N = 29): BL (N = 24), PMBCL (N = 5); HIV positive patients (N = 12): BL (N = 8), and DLBCL (N = 4). Among those with BL, majority had stage III/IV disease (N = 21/32, 65%). At the completion of planned therapy, 33 had achieved CR (81%). One patient died due to toxicity. The actuarial EFS and OS at 2 years were 80 and 77% respectively for all patients. The OS at 2 years was 100% for PMBCL, 80% for BL and 50% for HIV-positive DLBCL. Majority of the failures in BL were in patients with advanced disease. DA-EPOCH-R can be used in real-world setting and allows treatment of older patients with BL.     

Time course of the changes in right and left ventricle function and associated factors after transcatheter closure of atrial septal defects

Objective

The purpose of this study was to evaluate the changes in right ventricle (RV) and left ventricle (LV) function after transcatheter atrial septal defect (ASD) closure and to assess the influence of the age and the amount of shunt.

Design

Retrospective study

Patients

Fifty‐three adult patients who underwent transcatheter closure were enrolled, then divided into subgroups according to the age (< 40 years vs ≥ 40 years), and the amount of shunt flow (QpQs < 2.5 vs QpQs ≥ 2.5).

Outcome Measures

Two‐dimensional tissue Doppler imaging was performed in a four‐chamber view at the basal ventricular septum (VS) and tricuspid valve annulus (TVA) before and at 1 month and 6 months after closure. Myocardial velocities, the myocardial performance index (MPI), and isovolumic acceleration (IVA) were assessed.

Results

At the TVA, the MPI decreased slightly and then greatly increased at 6 months after closure (P = .002). The IVA improved in all patients (P < .001), and the E′/A′ ratio decreased, especially in the old age group (P = .031) and larger shunt group (P = .035). At the VS, S′ and the IVA decreased and had not recovered until 6 months in the old age (P = .02) and larger shunt (P = .02). The Qp/Qs showed a significant reverse correlation with a decrease in the E′/A′ at the TVA (r = ?0.37, P = .008), and age of patient was correlated with a decrease in the IVA at the VS (r = ?0.32, P = .019). The age at closure (β = ?0.36, P = .002), the Qp/Qs ratio (β = ?0.45, P = .01), and RV MPI changes (β = ?7.64, P < .001) were found to be associated factors with IVA decrease at the VS.

Conclusions

After ASD closure, RV global function might be impaired. In elderly patients and patients with a large shunt, impairment of LV contractility developed until 6 months after closure. Close long‐term observation is required after closure, especially in old‐age patients with a large shunt.