Neoadjuvant radiation therapy and its impact on complications after pancreaticoduodenectomy for pancreatic cancer: analysis of the American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP)

Objectives

This study investigated the impact of neoadjuvant radiation therapy (XRT) on postoperative outcomes following pancreaticoduodenectomy for pancreatic cancer.

Methods

The American College of Surgeons National Quality Improvement Program database was queried for the period 2005–2010 to assess complication rates following pancreaticoduodenectomy for pancreatic cancer. Two groups of patients were identified, comprising those who received neoadjuvant XRT and those who did not (control group).

Results

A total of 4416 patients were identified, including 200 in the XRT group and 4216 in the control group. There were differences in patient characteristics between the groups, including in age, hypertension and bilirubin level. Despite the fact that weight loss was more common, median operative time was longer (423 min versus 368 min; P < 0.001), and vascular reconstruction was more commonly required (20.5% versus 8.4%; P < 0.001) in the XRT group. In addition, the XRT group had a shorter median hospital stay than the control group (9 days versus 10 days; P = 0.005). Mortality (3.0% versus 2.7%; P = 0.818) and morbidity (40.5% versus 37.6%; P = 0.404) rates were not influenced by neoadjuvant XRT. Blood transfusion rates were increased in the XRT group (13.0% versus 7.4%; P = 0.003). Severe complications were influenced by age >70 years, American Society of Anesthesiologists (ASA) class >2, preoperative sepsis, dyspnoea, weight loss, impaired functional status, peripheral vascular disease and operative time of >8 h.

Conclusions

Neoadjuvant XRT is not associated with an increase in complications after pancreaticoduodenectomy.     

Acute pulmonary vein reconnection is a predictor of atrial fibrillation recurrence following pulmonary vein isolation

Purpose

Arrhythmia recurrence following pulmonary vein isolation (PVI) occurs predominantly due to the reconnection of previously isolated pulmonary veins (PVs). The prognostic implications of detection and treatment of acute PV reconnection are not well understood. We aim to examine the prognostic significance of acute PV reconnection on arrhythmia recurrence at 1 year following PVI.

Methods

This prospective study included 44 patients (22 men, 60?±?7 years) who underwent index PVI procedure for treatment of atrial fibrillation (AF). Acute PV reconnection and/or dormant PV conduction were assessed sequentially in response to a 30-min waiting period, intravenous isoproterenol infusion and/or adenosine. All cases of acute PV reconnection and/or dormant conduction were successfully targeted with additional ablation.

Results

Freedom from AF at 1 year was 75 % (83.3 % in paroxysmal and 65 % in persistent AF, p?=?ns). Acute PV reconnection and/or dormant conduction were evident in 16 of 44 patients (36.3 %). AF recurrence was documented in eight of 16 patients with, but only in three of 28 patients without acute reconnection (p?=?0.009). Three patients underwent a redo procedure, all from the group of patients with acute PV reconnection. In a multivariate model, acute PV reconnection was a strong independent predictor of arrhythmia recurrence (hazards ratio [HR], 6.36; 95 % confidence interval [CI], 1.12–31.6).

Conclusion

Identification of acute PV reconnection, even when successfully targeted, is a strong predictor of arrhythmia recurrence following PVI.     

Safety and efficacy of sofosbuvir/velpatasvir/voxilaprevir in post-liver transplant patients with previous direct-acting antiviral failure: Six case reports

BACKGROUNDDirect-acting antiviral (DAA) therapy regimens are highly effective at eliminating hepatitis C virus (HCV) infection but rates of sustained virologic response (SVR) are lower in patients with decompensated cirrhosis or hepatocellular carcinoma. Since many of these patients will be referred for liver transplant, they will require retreatment after transplantation. Sofosbuvir/velpatasvir/voxilaprevir (SOF/VEL/VOX) is recommended by guidelines as the preferred regimen to treat HCV in DAA-experienced patients following liver transplant however there is limited data.CASE SUMMARYWe present the cases of six liver transplant recipients who had previous treatment failure with sofosbuvir-based DAA therapy prior to transplantation and who then received SOF/VEL/VOX after transplant.CONCLUSIONThis case series demonstrate the real-world efficacy and safety of SOF/VEL/VOX in the post liver transplant setting. Treatment was successful with all patients achieving SVR, it was well tolerated, and there were minimal drug-drug interactions with their immunosuppressants.     

Phosphoglucan-bound structure of starch phosphatase Starch Excess4 reveals the mechanism for C6 specificity

Plants use the insoluble polyglucan starch as their primary glucose storage molecule. Reversible phosphorylation, at the C6 and C3 positions of glucose moieties, is the only known natural modification of starch and is the key regulatory mechanism controlling its diurnal breakdown in plant leaves. The glucan phosphatase Starch Excess4 (SEX4) is a position-specific starch phosphatase that is essential for reversible starch phosphorylation; its absence leads to a dramatic accumulation of starch in Arabidopsis, but the basis for its function is unknown. Here we describe the crystal structure of SEX4 bound to maltoheptaose and phosphate to a resolution of 1.65 Å. SEX4 binds maltoheptaose via a continuous binding pocket and active site that spans both the carbohydrate-binding module (CBM) and the dual-specificity phosphatase (DSP) domain. This extended interface is composed of aromatic and hydrophilic residues that form a specific glucan-interacting platform. SEX4 contains a uniquely adapted DSP active site that accommodates a glucan polymer and is responsible for positioning maltoheptaose in a C6-specific orientation. We identified two DSP domain residues that are responsible for SEX4 site-specific activity and, using these insights, we engineered a SEX4 double mutant that completely reversed specificity from the C6 to the C3 position. Our data demonstrate that the two domains act in consort, with the CBM primarily responsible for engaging glucan chains, whereas the DSP integrates them in the catalytic site for position-specific dephosphorylation. These data provide important insights into the structural basis of glucan phosphatase site-specific activity and open new avenues for their biotechnological utilization.Starch is the primary carbohydrate storage molecule in plants and is an essential constituent of human and animal diets. Starch granules are composed of the glucose homopolymers amylose (10–25%) and amylopectin (75–90%) (1, 2). Amylose is a linear molecule formed from α-1,4-glycosidic–linked chains, whereas amylopectin is formed from α-1,4-glycosidic–linked chains with α-1,6-glycosidic–linked branches (3, 4). Adjacent amylopectin chains interact to form double helices that cause starch granules to be water insoluble, which is an essential feature for its function as a glucose storage molecule (1, 3, 5). However, the outer granular surface of transitory starch must be solubilized during nonphotosynthetic periods so that glycolytic enzymes can access and degrade starch glucans and meet the metabolic needs of the plant (6, 7). Plants regulate the solubility of the starch granular surface via reversible starch phosphorylation that results in a cyclic degradative process: phosphorylation by dikinases, degradation by starch hydrolyzing amylases, and dephosphorylation by phosphatases (1, 8–11). Phosphorylation of amylopectin chains causes helical unwinding and local solubilization of the outer starch granule (12–14). The local solubilization and helix unwinding permits degradation of surface, linear α-1,4 glucan chains by β-amylase, which sequentially removes maltosyl units from the nonreducing end (1, 8, 15). Although glucan phosphorylation of the starch surface is necessary for degradation, the removal of these phosphate groups is required because β-amylase is unable to degrade past the phosphate (6, 15–17). Therefore, glucan phosphatases must release phosphate from starch to reset the degradation cycle, allowing processive starch degradation (8, 16).Recent studies have established that plants use a two-enzyme system for both starch phosphorylation and dephosphorylation. α-Glucan water dikinase phosphorylates the hydroxyl group of starch glucose at the C6 position. This event triggers phosphorylation of the hydroxyl group at the C3 position by phosphoglucan water dikinase (18–21). Similarly, two glucan phosphatases release phosphate from starch. Starch Excess4 (SEX4) preferentially dephosphorylates the C6 position of starch glucose and Like Sex Four2 (LSF2) exclusively dephosphorylates the C3 position (22–26). SEX4 activity is essential for starch catabolism and its mutation in Arabidopsis leads to an excess of leaf starch, a decrease in plant growth, and an accumulation of soluble phosphoglucans produced by the activity of α-amylase 3 and the debranching enzyme isoamylase 3 (16, 25, 27). Conversely, lsf2 mutant Arabidopsis plants display normal levels of leaf starch and plant growth, but the starch contains increased levels of C3-phosphate (22). The difference in plant vitality between sex4 and lsf2 mutants is likely due to SEX4 possessing some compensatory C3-position phosphatase activity (22). Cumulatively, the process of reversible phosphorylation requires the concerted activity of dikinases and phosphatases with SEX4 activity being essential for normal patterns of starch metabolism and plant growth.Glucan phosphatases are members of the protein tyrosine phosphatase (PTP) superfamily characterized by a conserved Cx₅R catalytic motif (24, 28, 29). The glucan phosphatases belong to a heterogeneous subset of PTPs called dual-specificity phosphatases (DSPs), with some DSPs dephosphorylating p-Tyr and p-Ser/Thr residues of proteinaceous substrates and other DSPs dephosphorylating lipids, nucleic acids, or glucans (30–32). In addition to SEX4 and LSF2, a glucan phosphatase called laforin has been identified that dephosphorylates glycogen and influences its solubility in vertebrates (24, 33, 34). Loss of laforin function in humans results in a fatal, neurodegenerative epilepsy called Lafora disease (35–37). Due to their critical function in complex carbohydrate metabolism, understanding the structural basis of glucan phosphatase activity is of particular interest. Toward this goal, we previously determined the ligand-free structure of SEX4 and identified an extensive interdomain interface between its DSP domain and carbohydrate-binding module (CBM) that is maintained in part by a previously unrecognized C-terminal (CT) motif (38). However, the structural mechanism for domain coupling, glucan interaction, and specific C6 dephosphorylation in SEX4 activity is unclear.Starch granule solubilization depends on phosphorylation of starch glucose on the hydroxyl group at both the C6 and C3 positions (6, 13). These phosphorylation events are critical for normal transitory starch degradation, but also directly impact the melting temperature, viscosity, and hydration of starch in industrial settings (39, 40). Developing a means to manipulate starch phosphorylation patterns via enzymatic modification is relevant to agricultural and industrial applications that use starch as a feedstock (9, 12, 14, 41). Therefore, understanding the basis for the site specificity of glucan phosphatases is of particular interest. We recently determined the structure of LSF2 with a glucan bound in a C3-specific orientation and identified unique noncatalytic surface-binding sites (SBSs) not found in other glucan phosphatases (26). SEX4 lacks SBSs and preferentially dephosphorylates the C6 position. The present study was designed to define the fundamental basis for SEX4 substrate binding and understand preferential C6-position specificity in SEX4.Herein, we elucidate the structural mechanism of SEX4-specific activity by determining the structure of SEX4 bound to the phosphoglucan products maltoheptaose and phosphate. SEX4 engages glucan chains via an extended interface of aromatic and hydrophilic residues that spans the CBM and DSP domains. Moreover, the SEX4 CBM is primarily responsible for glucan binding whereas the SEX4 DSP active site is uniquely adapted to engage the phosphoglucan substrate, positioning it in a C6-specific orientation. Structure-guided mutagenesis of DSP active-site residues resulted in a complete reversal from C6 to preferential C3 dephosphorylation by SEX4. Cumulatively, this study establishes the molecular basis for both SEX4 substrate engagement and SEX4 specificity and provides a method for engineering glucan phosphatase activity with modified site specificity.     

Spatially robust estimates of biological nitrogen (N) fixation imply substantial human alteration of the tropical N cycle

Biological nitrogen fixation (BNF) is the largest natural source of exogenous nitrogen (N) to unmanaged ecosystems and also the primary baseline against which anthropogenic changes to the N cycle are measured. Rates of BNF in tropical rainforest are thought to be among the highest on Earth, but they are notoriously difficult to quantify and are based on little empirical data. We adapted a sampling strategy from community ecology to generate spatial estimates of symbiotic and free-living BNF in secondary and primary forest sites that span a typical range of tropical forest legume abundance. Although total BNF was higher in secondary than primary forest, overall rates were roughly five times lower than previous estimates for the tropical forest biome. We found strong correlations between symbiotic BNF and legume abundance, but we also show that spatially free-living BNF often exceeds symbiotic inputs. Our results suggest that BNF in tropical forest has been overestimated, and our data are consistent with a recent top-down estimate of global BNF that implied but did not measure low tropical BNF rates. Finally, comparing tropical BNF within the historical area of tropical rainforest with current anthropogenic N inputs indicates that humans have already at least doubled reactive N inputs to the tropical forest biome, a far greater change than previously thought. Because N inputs are increasing faster in the tropics than anywhere on Earth, both the proportion and the effects of human N enrichment are likely to grow in the future.Over the last few decades, humans have dramatically altered the global nitrogen (N) cycle (1–3). Three main processes—Haber–Bosch fixation of atmospheric N₂, widespread cultivation of leguminous N-fixing crops, and incidental N fixation during fossil fuel combustion—collectively add more reactive N to the biosphere each year than all natural processes combined (2). Although human perturbation of the N cycle has brought substantial benefits to society (most notably, an increase in crop production) (4), it has also had a number of negative effects on both ecosystems (5, 6) and people (7).Although humanity’s large imprint on the global N cycle is clear, quantifying the extent of anthropogenic changes depends, in large part, on establishing baseline estimates of nonanthropogenic N inputs (1, 8, 9). Before recent human activities, biological N fixation (BNF) was the largest source of new N to the biosphere (9). Terrestrial BNF has been particularly challenging to quantify, because it displays high spatial and temporal heterogeneity at local scales, it arises from both symbiotic associations between bacteria and plants as well as free-living microorganisms (e.g., in leaf litter and soil) (10), and high atmospheric concentrations of N₂ make direct flux measurements unfeasible. Consequently, spatial estimates of BNF have always been highly uncertain (11), and global rate estimates have fallen precipitously in the last 15 y (from 100–290 to ∼44 Tg N y⁻¹) (9). This decline in BNF implies an increase in the relative magnitude of anthropogenic N inputs from 100–150% to 190–470% of BNF (9).Historically, the largest anthropogenic changes to the N cycle have occurred in the northern temperate zone: first throughout the United States and western Europe and more recently, in China (12, 13). Large-scale estimates of BNF in natural ecosystems in these regions are consistently low (11), leading some to conclude that anthropogenic N inputs in the northern temperate zone exceed naturally occurring BNF and preindustrial atmospheric N deposition by an order of magnitude or more (1, 14). By contrast, the highest rates of naturally occurring BNF have been thought to occur in the evergreen lowland tropical rainforest biome (11), implying that, on a regional basis, human alteration of the tropical N cycle has been comparatively modest. However, in recent years, the tropics have seen some of the most dramatic increases in anthropogenic N inputs of any region on Earth—a trend that is likely to continue (2, 6, 13). Anthropogenic N inputs are increasing in tropical regions, primarily because of increasing fossil fuel combustion (13) and expanding high-N-input agriculture for both food and biofuels (6). These anthropogenic N inputs are having a measurable effect on tropical ecosystems (15). However, understanding and forecasting the effects of anthropogenic N depend, in part, on accurate estimates of BNF in lowland tropical rainforest.Unfortunately, the paradigm that the tropics have high rates of BNF is based on a paucity of evidence and several tenuous assumptions. For example, an early global synthesis of terrestrial BNF (11)—which included contributions from both symbiotic and free-living sources—included only one measured estimate of symbiotic BNF from tropical forest (16 kg N ha⁻¹ y⁻¹) (16). That single estimate, scaled over thousands of square kilometers, represented the only direct evidence of high tropical BNF rates available at that time (Fig. 1). Subsequent modeled estimates (17) that indirectly estimated BNF have reinforced the notion that tropical BNF rates are high and dominated by the symbiotic form of fixation (Fig. 1). Such high estimates of symbiotic BNF are consistent with the large number of leguminous trees in tropical forest (18–20). However, many legume species do not form N-fixing nodules (21), and of those species that do, nodulation in individuals varies with soil nutrient status, N demand, and tree age (22). Several recent analyses (10, 22–24) indicate lower tropical forest BNF and suggest that symbiotic BNF may not be as important to total BNF as previously thought (Fig. 1), although few studies have simultaneously measured symbiotic and free-living BNF.Open in a separate windowFig. 1.Previous estimates of BNF in tropical rainforest and BNF measured in this study. Percentages indicate the proportion of total BNF from symbiotic BNF. Cleveland et al. 1999 A (11) is a literature database-derived estimate of tropical forest BNF; Cleveland et al. 1999 B (11) is a modeled estimate of BNF based on the correlation between net primary productivity (NPP) and BNF derived with remotely sensed NPP and evergreen broadleaved forest (EBF) land cover classification. Central estimates and variance for Cleveland et al., 1999 A (11) and Reed et al. 2011 (10) represent the low, central, and high data-based estimates of BNF assuming 5%, 15%, and 15% legume cover, respectively. Central estimates and variance for Wang and Houlton 2009 (17) represent the modeled mean and SD of BNF predicted for the EBF biome. Central estimates and variance for Cleveland et al. 2010 (23) represent the low, central, and high estimates of symbiotic BNF plus free-living BNF or modeled BNF plus free-living BNF. Central estimates and variance for BNF in the four forest ages measured here (primary, 5–15 y, 15–30 y, and 30–50 y) represent means ± 1 SD (n = 3). Our estimate of BNF in a dynamic primary forest (gap dynamics) lacks SD, because it consisted of only two measurements: low and high estimates of forest turnover times equal to 150 and 75 y, respectively.There is also a sound theoretical basis for questioning high estimates of BNF in tropical forest. Namely, high concentrations of soil N in the legume-rich tropics create something of a paradox. Although BNF could create N-rich conditions, the substantial energetic cost of BNF means—and some data show—that BNF should be suppressed under high N availability in primary forests (25). Because of high rates of net primary productivity and high N demand in secondary forests (26, 27), regenerating canopy gaps or abandoned agricultural land may have higher rates of BNF than late-successional forest ecosystems (26).Resolving the uncertainty in the tropical (and global) N cycle requires that we overcome the enduring challenge of quantifying BNF in any ecosystem. How do we estimate large-scale rates of a process that displays extreme spatial heterogeneity at local scales? Whether using acetylene reduction assays, ¹⁵N tracer incubations, or the ¹⁵N natural abundance method, most past approaches to empirically estimate symbiotic BNF have relied on spatial extrapolations of BNF rates measured at the level of individual trees. Typically, such extrapolations are based on legume abundance (e.g., percent cover) and make species- or genera-level assumptions about nodulation status of putative N fixers. Here, we applied a method commonly used by community ecologists to measure rare species abundances—stratified adaptive cluster sampling (SACS) (28)—to measure symbiotic BNF. This approach could be used in any ecosystem, and in contrast to other methods, SACS generates unbiased estimates of mean symbiotic BNF (independent of legume abundance) and can more robustly capture the irregular distribution of nodules on the landscape. We simultaneously measured symbiotic and free-living BNF multiple times over the course of 1 y to generate spatially explicit rates of BNF inputs in primary and secondary (5–50 y old) lowland tropical forest in Costa Rica and then used the understanding gained from those estimates to revisit estimates of BNF and anthropogenic N inputs in the tropical forest biome.     

Bcl-2, Bcl-x(L), and Bcl-w are not equivalent targets of ABT-737 and navitoclax (ABT-263) in lymphoid and leukemic cells

The BH3-mimetic ABT-737 and an orally bioavailable compound of the same class, navitoclax (ABT-263), have shown promising antitumor efficacy in preclinical and early clinical studies. Although both drugs avidly bind Bcl-2, Bcl-x(L), and Bcl-w in vitro, we find that Bcl-2 is the critical target in vivo, suggesting that patients with tumors overexpressing Bcl-2 will probably benefit. In human non-Hodgkin lymphomas, high expression of Bcl-2 but not Bcl-x(L) predicted sensitivity to ABT-263. Moreover, we show that increasing Bcl-2 sensitized normal and transformed lymphoid cells to ABT-737 by elevating proapoptotic Bim. In striking contrast, increasing Bcl-x(L) or Bcl-w conferred robust resistance to ABT-737, despite also increasing Bim. Cell-based protein redistribution assays unexpectedly revealed that ABT-737 disrupts Bcl-2/Bim complexes more readily than Bcl-x(L)/Bim or Bcl-w/Bim complexes. These results have profound implications for how BH3-mimetics induce apoptosis and how the use of these compounds can be optimized for treating lymphoid malignancies.