A new species of Bolitoglossa (Caudata: Plethodontidae) of the Bolitoglossa franklini group from an isolated cloud forest in northern Guatemala.

We describe Bolitoglossa qeqom sp. nov. from an isolated cloud forest in Cerro Guachmalén, Alta Verapaz, Guatemala, based on multiple lines of evidence (morphological, molecular, and biogeographic data). This region comprises a mountain ridge without previous herpetological surveys. The new species is a large salamander with uniform purplish-black coloration and is distinguished by having relatively long legs with only one costal groove between adpressed limbs, numerous maxillary teeth, few vomerine teeth, only one phalange free of webbing in digit III of feet, and a relatively short tail. It is geographically closest to its sister clade of B. lincolni + B. franklini and the xeric Chixoy river canyon appears to be the major biogeographic barrier that isolated the new taxon. The cloud forest inhabited by this species has undergone severe habitat destruction in the region and land conservation actions are urgent.

Age stratified, matched comparison of unicompartmental and total knee replacement.

BACKGROUND: Unicompartmental knee replacement (UKR) tends to provide better function but has a higher revision rate than total knee replacement (TKR). The aim was to determine if this occurred in all age groups. METHODS: Two large, non-registry, prospective cohorts with median 10-year follow-up (2252 TKR, 1000 medial UKR) were identified. All UKR met recommended indications. TKR with an inappropriate disease pattern for medial UKR were excluded. Knees were propensity score-matched within age-strata (<60 years at operation, 60 to <75, 75+) and compared using Oxford Knee Score (OKS), Kaplan-Meier revision rates and a composite failure, defined as any of revision, reoperation or no improvement in OKS. RESULTS: One thousand five hundred and eighty-two TKR and UKR were matched. Results are reported TKR vs UKR for ages <60, 60 to <75 and 75+. Median 10-year OKS were 33 vs 45 (p < 0.001), 36 vs 42 (p < 0.001) and 36 vs 38 (p = 0.25). Ten-year revision rates were 11% vs 7%, 5% vs 5%, and 5% vs 10%, (none significant). The composite failures occurred 8%, 5% and 5% more frequently with TKR than UKR (none significant). CONCLUSIONS: In this matched study UKR provided better functional outcomes in all age groups, particularly the young, and provided substantially more excellent outcomes. Although in older groups TKR tended to have a lower revision rate, in the young UKR had a lower revision rate. This was surprising and was perhaps because in this study UKR was, as recommended, only used for bone-on-bone arthritis, whereas in young patients it is widely used for early arthritis, which is associated with a high failure rate. This study supports the use of UKR with recommended indications, in all age groups.

Palliative Lung Radiotherapy: Higher Dose Leads to Improved Survival?

AIMS: Choosing the optimal palliative lung radiotherapy regimen is challenging. Guidance from The Royal College of Radiologists recommends treatment stratification based on performance status, but evidence suggests that higher radiotherapy doses may be associated with survival benefits. The aim of this study was to investigate the effects of fractionation regimen and additional factors on the survival of palliative lung cancer radiotherapy patients. MATERIALS AND METHODS: A retrospective univariable (n = 925) and multivariable (n = 422) survival analysis of the prognostic significance of baseline patient characteristics and treatment prescription was carried out on patients with non-small cell and small cell lung cancer treated with palliative lung radiotherapy. The covariates investigated included: gender, age, performance status, histology, comorbidities, stage, tumour location, tumour side, smoking status, pack year history, primary radiotherapy technique and fractionation scheme. The overall mortality rate at 30 and 90 days of treatment was calculated. RESULTS: Univariable analysis revealed that performance status (P < 0.001), fractionation scheme (P < 0.001), comorbidities (P = 0.02), small cell histology (P = 0.02), 'lifelong never' smoking status (P = 0.01) and gender (P = 0.06) were associated with survival. Upon multivariable analysis, only better performance status (P = 0.01) and increased dose/fractionation regimens of up to 30 Gy/10 fractions (P < 0.001) were associated with increased survival. Eighty-five (9.2%) and 316 patients (34%) died within 30 and 90 days of treatment, respectively. CONCLUSION: In this retrospective single-centre analysis of palliative lung radiotherapy, increased total dose (up to and including 30 Gy/10 fractions) was associated with better survival regardless of performance status.

Lifetime revision risk for medial unicompartmental knee replacement is lower than expected.

PURPOSE: Unicompartmental knee replacement (UKR) is widely considered to be a pre-total knee replacement (TKR) particularly in the young. The implication of this is that it is sensible to do a UKR, even though it will be revised at some stage, as it will delay the need for a TKR. The chance of a UKR being revised during a patient's life time has not previously been calculated. The aim of this study was to estimate this lifetime revision risks for patients of different ages undergoing UKR. METHODS: Calculations were based on data from a designer series of 1000 medial Oxford UKR with mean 10-year follow up. These UKR were implanted for the recommended indications using the recommended surgical technique. Parametric survival models were developed for patients of different ages based on observed data, and were extrapolated using a Markov model to estimate lifetime revision risk. RESULTS: The estimated lifetime revision risk reduced with increasing age at surgery. Lifetime revision risk at age 55 was 15% (95% CI 12-19), at 65 it was 11% (8-13), at 75 it was 7% (5-9), and at 85 it was 4% (3-5). CONCLUSION: Provided UKR is used appropriately, the lifetime revision risk is markedly lower than expected. UKR should be considered to be a definitive knee replacement rather than a Pre-TKR even in the young. These lifetime estimates, alongside established benefits for UKR in speed of recovery, morbidity, mortality and function, can be discussed with appropriate patients when considering whether to implant a UKR or TKR. LEVEL OF EVIDENCE: III.

Advanced microscopy analysis of the micro-nanoscale architecture of human menisci.

The complex inhomogeneous architecture of the human meniscal tissue at the micro and nano scale in the absence of artefacts introduced by sample treatments has not yet been fully revealed. The knowledge of the internal structure organization is essential to understand the mechanical functionality of the meniscus and its relationship with the tissue's complex structure. In this work, we investigated human meniscal tissue structure using up-to-date non-invasive imaging techniques, based on multiphoton fluorescence and quantitative second harmonic generation microscopy complemented with Environmental Scanning Electron Microscopy measurements. Observations on 50 meniscal samples extracted from 6 human menisci (3 lateral and 3 medial) revealed fundamental features of structural morphology and allowed us to quantitatively describe the 3D organisation of elastin and collagen fibres bundles. 3D regular waves of collagen bundles are arranged in "honeycomb-like" cells that are comprised of pores surrounded by the collagen and elastin network at the micro-scale. This type of arrangement propagates from macro to the nanoscale.

Mid- to long-term function and implant survival of ACL reconstruction and medial Oxford UKR.

PURPOSE: The purpose of this study was to describe mid- to long-term outcomes of anterior cruciate ligament (ACL) reconstruction with simultaneous or staged medial unicompartmental knee replacement (UKR), and compare outcomes between (1) young patients aged younger than 55 at surgery and those older, (2) those with long-term follow-up greater than 10 years, (3) cemented and cementless UKR, and (4) compare outcomes to those with an intact ACL. PATIENTS AND METHODS: We identified knees with staged or simultaneous ACL reconstruction and medial UKR from a prospectively followed designer UKR cohort, and describe mean Oxford Knee Score (OKS), mean Tegner activity score and Kaplan-Meier survival estimates. We matched these knees to ACL-intact knees. RESULTS: Seventy-six consecutive UKR with staged or simultaneous ACL reconstruction were identified with mean six-year follow-up (range 1-15). There was significant improvement in OKS and Tegner score with surgery. At most recent follow-up, OKS was 41.0 (range 11 to 48), and Tegner score 3.6 (0 to 8). There were three revisions occurring at a mean of five years post-operatively. The five-, 10- and 15-year survival estimates were 97% (95% confidence interval [CI] 93-100), 92% (83-100), and 92% (83-100). There was no difference in functional scores or implant survival in young patients, those with long-term follow-up (>10 years), those with cementless fixation, or when compared to ACL intact knees. CONCLUSION: These results demonstrate excellent mid- to long-term function and survival of selected patients who have undergone ACL reconstruction and medial UKR. Their outcome was similar to those with intact ACLs.

Functional Outcome and Revision Rate Are Independent of Limb Alignment Following Oxford Medial Unicompartmental Knee Replacement.

BACKGROUND: There is controversy about optimal limb alignment following knee replacement. An aim of using Oxford medial unicompartmental knee replacement (UKR) implants is to accurately restore normal ligament tension in the knee, thereby restoring normal kinematics. This return to normal tension typically results in a return to prearthritic alignment, which is frequently varus. The aim of this study was to investigate the relationship between postoperative limb alignment and postoperative patient-reported outcome and implant revision rate. METHODS: We used a consecutive cohort of 891 knees with cemented Oxford medial UKR implants with a mean 10-year follow-up and recorded alignment. We grouped knees according to postoperative mechanical alignment as marked varus (estimated at 10°), mild varus (estimated at 5°), neutral, and valgus. The mean Oxford Knee Score (OKS) was calculated at 5 and 10 years postoperatively. Revision risk was assessed by survival analysis and component-time incidence rates. RESULTS: Postoperatively, 67 (8%) of the 891 knees were in marked varus; 308 (35%), in mild varus; 508 (57%), in neutral; and 8 (1%), in valgus. The valgus group (8 knees) was too small for further analysis. The mean OKS (and standard deviation [SD]) at 10 years postoperatively was 41.7 ± 7 for marked varus, 40.5 ± 8 for mild varus, and 39.4 ± 9 for neutral alignment (p = 0.28). At 10 years, 92%, 85%, and 76% achieved a good or excellent OKS outcome, respectively (p = 0.02). Twelve-year survival rates were 93.3% for marked varus, 93.2% for mild varus, and 93.6% for neutral alignment, respectively (p = 0.53). Revision incidence rates per 100 component-years were 0.49 (95% confidence interval [CI], 0.2 to 1.5), 0.36 (95% CI, 0.2 to 0.7), and 0.54 (95% CI, 0.4 to 0.8), respectively, and were not significantly different (p = 0.53). CONCLUSIONS: Marked postoperative varus mechanical alignment of an estimated 10° was present in 8%, and mild varus of about 5° was present in 35%. Increasing varus alignment was associated with an increasing percentage of good or excellent OKS outcomes, but otherwise there were no significant differences between alignment groups in patient-reported outcome or revision rate. These data support the standard operative technique for the Oxford UKR, which aims to restore ligament tension and therefore prearthritic alignment rather than neutral mechanical alignment. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Intraosseous pressure during loading and with vascular occlusion in an animal model.

OBJECTIVES: We studied subchondral intraosseous pressure (IOP) in an animal model during loading, and with vascular occlusion. We explored bone compartmentalization by saline injection. MATERIALS AND METHODS: Needles were placed in the femoral condyle and proximal tibia of five anaesthetized rabbits and connected to pressure recorders. The limb was loaded with and without proximal vascular occlusion. An additional subject had simultaneous triple recordings at the femoral head, femoral condyle and proximal tibia. In a further subject, saline injections at three sites were carried out in turn. RESULTS: Loading alone caused a rise in subchondral IOP from 11.7 mmHg (sd 7.1) to 17.9 mmHg (sd 8.1; p < 0.0002). During arterial occlusion, IOP fell to 5.3 mmHg (sd 4.1), then with loading there was a small rise to 7.6 mmHg (sd 4.5; p < 0.002). During venous occlusion, IOP rose to 20.2 mmHg (sd 5.8), and with loading there was a further rise to 26.3 mmHg (sd 6.3; p < 0.003). The effects were present at three different sites along the limb simultaneously. Saline injections showed pressure transmitted throughout the length of the femur but not across the knee joint. CONCLUSION: This is the first study to report changes in IOP in vivo during loading and with combinations of vascular occlusion and loading. Intraosseous pressure is not a constant. It is reduced during proximal arterial occlusion and increased with proximal venous occlusion. Whatever the perfusion state, in vivo load is transferred partly by hydraulic pressure. We propose that joints act as hydraulic pressure barriers. An understanding of subchondral physiology may be important in understanding osteoarthritis and other bone diseases.Cite this article: M. Beverly, S. Mellon, J. A. Kennedy, D. W. Murray. Intraosseous pressure during loading and with vascular occlusion in an animal model. Bone Joint Res 2018;7:511-516. DOI: 10.1302/2046-3758.78.BJR-2017-0343.R2.

Survival following operative management of gastric linitis plastica compared with non-operative management.

INTRODUCTION Linitis plastica (LP) is a particular subtype of diffuse gastric cancer and is thought to have a very poor prognosis. The operative approach in patients with LP has historically been questioned because of the poor outcomes. The aim of this study was to determine the current outcomes in LP patients who undergo radical resection. METHODS Patients with a new diagnosis of diffuse gastric adenocarcinoma between 2006 and 2010 were identified from a regional pathology database. LP was diagnosed based on histological, radiological and endoscopic findings. The patients' health records were analysed retrospectively and mortality data obtained from a regional cancer registry. The primary outcome assessed was overall survival. RESULTS Overall, 273 patients with diffuse gastric cancer were identified; 54 of these were diagnosed with LP. In the LP cohort, 17 patients underwent resection compared with 95 of the 219 patients in the non-LP group. The median survival following resection in patients with LP was 16.7 months (95% confidence interval [CI]: 8.3-25.1) while in LP patients who did not have surgery it was 3.6 months (95% CI: 2.2-4.9 months) (p<0.001). There was no significant difference in survival following resection between those with LP and those with non-LP diffuse gastric adenocarcinoma (median: 23.9 months, 95% CI: 15.8-32.1 months) (p=0.331). CONCLUSIONS Survival following resection in patients with LP is not significantly different to that in those with non-LP diffuse gastric cancer. A preoperative diagnosis of LP should not be a reason for denying radical treatment and such individuals should be managed in the same way as any other patient with diffuse gastric cancer.

The current role of staging laparoscopy in oesophagogastric cancer.

INTRODUCTION: Oesophagogastric cancers are known to spread rapidly to locoregional lymph nodes and by transcoelomic spread to the peritoneal cavity. Staging laparoscopy combined with peritoneal cytology can detect advanced disease that may not be apparent on other staging investigations. The aim of this study was to determine the current value of staging laparoscopy and peritoneal cytology in light of the ubiquitous use of computed tomography in all oesophagogastric cancers and the addition of positron emission tomography in oesophageal cancer. METHODS: All patients undergoing staging laparoscopy for distal oesophageal or gastric cancer between March 2007 and August 2013 were identified from a prospectively maintained database. Demographic details, preoperative staging, staging laparoscopy findings, cytology and histopathology results were analysed. RESULTS: A total of 317 patients were identified: 159 (50.1%) had gastric adenocarcinoma, 136 (43.0%) oesophageal adenocarcinoma and 22 (6.9%) oesophageal squamous carcinoma. Staging laparoscopy revealed macroscopic metastases in 36 patients (22.6%) with gastric adenocarcinoma and 16 patients (11.8%) with oesophageal adenocarcinoma. Positive peritoneal cytology in the absence of macroscopic peritoneal metastases was identified in a further five patients with gastric adenocarcinoma and six patients with oesophageal adenocarcinoma. There was no significant difference in survival between patients with macroscopic peritoneal disease and those with positive peritoneal cytology (p=0.219). CONCLUSIONS: Staging laparoscopy and peritoneal cytology should be performed routinely in the staging of distal oesophageal and gastric cancers where other investigations indicate resectability. Currently, in our opinion, patients with positive peritoneal cytology should not be treated with curative intent.

Well-differentiated papillary mesothelioma: peritoneal implants are not always metastases in the presence of cancer.

INTRODUCTION: Well-differentiated papillary mesothelioma is a rare condition that can cause diagnostic uncertainty. Its appearance resembles metastatic deposits and in the presence of a known primary can often be confused for such. CASE PRESENTATION: In this case, we present a patient who had WDPM in the presence of gastric cancer and emphasise the difficulties in making the diagnosis. CONCLUSION: We highlight the need for a high index of suspicion, biopsies at staging laparoscopy and undertake a review of the literature regarding this uncommon condition.

Effect of triggering and entrapment on tendon gliding properties following digital flexor tendon laceration: in vitro study on turkey tendon.

The optimal management of partial flexor tendon laceration is controversial and remains a clinical challenge. Abnormal tendon gliding (triggering and entrapment) was assessed at the A2 pulley in 40 turkey tendons in three groups: intact, partially divided (palmar or lateral), and trimmed. Testing was of gliding resistance and friction coefficient at 30° and 70° of flexion, loaded with 2 and 4 N. We observed for triggering and entrapment. The changes in gliding properties were compared and analysed using Wilcoxon matched pair testing. A significant difference was found in the change in gliding properties of intact to lacerated and lacerated to trimmed tendons and between tendons that glided normally compared with those exhibiting triggering or entrapment. This suggests that palmar and lateral lacerations which, through clinical examination and visualization, are found to glide normally should be treated with early mobilization. However, partial lacerations that exhibit triggering or entrapment should be trimmed.

Safety and efficacy of 188-rhenium-labeled antibody to melanin in patients with metastatic melanoma.

There is a need for effective "broad spectrum" therapies for metastatic melanoma which would be suitable for all patients. The objectives of Phase Ia/Ib studies were to evaluate the safety, pharmacokinetics, dosimetry, and antitumor activity of (188)Re-6D2, a 188-Rhenium-labeled antibody to melanin. Stage IIIC/IV metastatic melanoma (MM) patients who failed standard therapies were enrolled in both studies. In Phase Ia, 10 mCi (188)Re-6D2 were given while unlabeled antibody preload was escalated. In Phase Ib, the dose of (188)Re-6D2 was escalated to 54 mCi. SPECT/CT revealed (188)Re-6D2 uptake in melanoma metastases. The mean effective half-life of (188)Re-6D2 was 12.4 h. Transient HAMA was observed in 9 patients. Six patients met the RECIST criteria for stable disease at 6 weeks. Two patients had durable disease stabilization for 14 weeks and one for 22 weeks. Median overall survival was 13 months with no dose-limiting toxicities. The data demonstrate that (188)Re-6D2 was well tolerated, localized in melanoma metastases, and had antitumor activity, thus warranting its further investigation in patients with metastatic melanoma.

Laparoscopic cardiomyotomy for achalasia: A single unit study.

AIMS: Achalasia is a rare incurable neuromuscular disorder of the oesophagus. A number of treatment options are available. We reviewed our results of laparoscopic cardiomyotomy over a 30 month period. METHODS: 18 patients with manometric features of achalasia underwent surgery between 2004 and 2006. Pre and postoperative weight and dysphagia scores were recorded (maximum score 45=normal, 0=complete dysphagia). Change in the Body Mass Index (BMI) was measured. Other symptoms (heartburn, epigastric pain, regurgitation, odynophagia and sleep disturbance) were scored on a 0-4 scale of increasing severity. RESULTS: At mean follow up of 16.2 months the mean dysphagia score was significantly improved from 7.5 to 33.9 (p<0.005). BMI was significantly increased from 22.3 to 25.8 kg/m(2) (p<0.05). Scores for heartburn, epigastric pain, regurgitation, odynophagia and sleep disturbance were also significantly improved. The average inpatient stay was 3.1 days and average operating time 111 minutes. One mucosal perforation occurred which was repaired intraoperatively. No patients required secondary operative intervention. CONCLUSIONS: Laparoscopic cardiomyotomy is a safe, highly effective, minimally invasive treatment for achalasia.

Investigating human leukemogenesis: from cell lines to in vivo models of human leukemia.

The hematopoietic system produces appropriate levels of blood cells over an individual's lifetime through a careful balance of differentiation, proliferation and self-renewal. The acquisition of genetic and epigenetic alterations leads to deregulation of these processes and the development of acute leukemias. A prerequisite to targeted therapies directed against these malignancies is a thorough understanding of the processes that subvert the normal developmental program of the hematopoietic system. This involves identifying the molecular lesions responsible for malignant transformation, their mechanisms of action and the cell type(s) in which they occur. Over the last 3 decades, significant progress has been made through the identification of recurrent genetic alterations and translocations in leukemic blast populations, and their subsequent functional characterization in cell lines and/or mouse models. Recently, primary human hematopoietic cells have emerged as a complementary means to characterize leukemic oncogenes. This approach enables the process of leukemogenesis to be precisely modeled in the appropriate cellular context: from primary human hematopoietic cells to leukemic stem cells capable of initiating disease in vivo. Here we review the model systems used to study leukemogenesis, and focus particularly on recent advances provided by in vitro and in vivo studies with primary human hematopoietic cells.

Wernicke's encephalopathy after laparoscopic cardiomyotomy for achalasia.

Achalasia is an incurable neuromuscular disorder of the esophagus, resulting from destruction of the esophageal myenteric plexus. This leads to aperistalsis and failure of the lower esophageal sphincter to relax after swallowing. Symptoms of achalasia are gradual in onset and include dysphagia, regurgitation, and weight loss. Severe malnutrition can ensue. Wernicke's encephalopathy (WE) is a serious, potentially fatal, neurologic disorder caused by thiamine deficiency (vitamin B(1)), classically described as presenting with a triad of ocular abnormalities, ataxia, and confusion. The incidence is uncertain, and many cases likely go unrecognized. It is usually diagnosed in the alcoholic population. We describe its onset after the successful surgical treatment of achalasia.

Expression of TEL-JAK2 in primary human hematopoietic cells drives erythropoietin-independent erythropoiesis and induces myelofibrosis in vivo.

Activation of JAK2 by chromosomal translocation or point mutation is a recurrent event in hematopoietic malignancies, including acute leukemias and myeloproliferative disorders. Although the effects of activated JAK2 signaling have been examined in cell lines and murine models, the functional consequences of deregulated JAK2 in the context of human hematopoietic cells are currently unknown. Here we report that expression of TEL-JAK2, a constitutively active variant of the JAK2 kinase, in lineage-depleted human umbilical cord blood cells results in erythropoietin-independent erythroid differentiation in vitro and induces the rapid development of myelofibrosis in an in vivo NOD/SCID xenotransplantation assay. These studies provide functional evidence that activated JAK2 signaling in primitive human hematopoietic cells is sufficient to drive key processes implicated in the pathophysiology of polycythemia vera and idiopathic myelofibrosis. Furthermore, they describe an in vivo model of myelofibrosis initiated with primary cells, highlighting the utility of the NOD/SCID xenotransplant system for the development of experimental models of human hematopoietic malignancies.

Beneficial clinical effects of perhexiline in patients with stable angina pectoris and acute coronary syndromes are associated with potentiation of platelet responsiveness to nitric oxide.

AIMS: To examine whether the prophylactic antianginal agent perhexiline potentiates platelet responsiveness to nitric oxide (NO) in patients with stable angina pectoris (SAP) and acute coronary syndromes (ACS: unstable angina pectoris or non-Q-wave myocardial infarction). METHODS AND RESULTS: Blood samples were obtained from patients before and after initiation of treatment with perhexiline. ADP-induced platelet aggregation and its inhibition by the NO donor sodium nitroprusside (SNP) were determined via impedance aggregometry in whole blood (WB) and platelet-rich plasma (PRP). Intraplatelet cGMP content was assayed by RIA, and superoxide (O(2)(-)) level by lucigenin-derived chemiluminescence. In patients with ACS not receiving perhexiline (n=12), platelet responsiveness to SNP did not vary significantly over the first 3 days post admission to hospital. Therapy with perhexiline for 3 days was associated with increases in SNP-induced inhibition of aggregation from 29+/-2% to 43+/-4% (n=50,P <0.001) in WB and from 20+/-5% to 42+/-7% (n=12, P<0.01) in PRP. Resolution of symptomatic ischaemia (n=39) was associated with significantly greater (P<0.01) increases than non-resolution (n=11). Similar increases in SNP responsiveness (P<0.001) occurred following institution of perhexiline therapy in patients with SAP (n=30), associated with a 85% decrease in anginal frequency. Treatment with perhexiline potentiated the cGMP-elevating effects of SNP in platelets (n=9,P =0.03). Although perhexiline did not alter whole blood O(2)(-) concentration ex vivo, it inhibited (P<0.01) O(2)(-) release from neutrophils in vitro. CONCLUSION: Perhexiline potentiates platelet responsiveness to NO both in SAP and ACS patients; in the latter group this improvement was predictive of resolution of ischaemic symptoms. The predominant mechanism of perhexiline effect is an increase in platelet cGMP responsiveness. Perhexiline also may reduce the potential for NO clearance by neutrophil-derived O(2)(-).

Composition of grape skin proanthocyanidins at different stages of berry development.

The composition of grape (Vitis vinifera L. cv. Shiraz) skin proanthocyanidins has been determined at different stages of berry development. Beginning approximately 3 weeks after fruit set and concluding at commercial ripeness, the composition of isolated skin proanthocyanidins was determined using the following analytical techniques: elemental analysis, UV-Vis absorption spectroscopy, reversed-phase HPLC after acid-catalysis in the presence of excess phloroglucinol, gel permeation chromatography, electrospray ionization mass spectrometry (ESI-MS), and (13)C NMR. On the basis of these analyses, berry development was correlated with an increase in proanthocyanidin mean degree of polymerization, an increase in the proportion of (-)-epigallocatechin extension subunits, and increases in the level of anthocyanins associated with the proanthocyanidin fraction. Additionally, data acquired from ESI-MS of the isolates following acid-catalysis in the presence of excess phloroglucinol is consistent with pectin-bound proanthocyanidins.