Phospholamban inhibits the cardiac calcium pump by interrupting an allosteric activation pathway.

Phospholamban (PLB) is a transmembrane micropeptide that regulates the sarcoplasmic reticulum Ca2+-ATPase (SERCA) in cardiac muscle, but the physical mechanism of this regulation remains poorly understood. PLB reduces the Ca2+ sensitivity of active SERCA, increasing the Ca2+ concentration required for pump cycling. However, PLB does not decrease Ca2+ binding to SERCA when ATP is absent, suggesting PLB does not inhibit SERCA Ca2+ affinity. The prevailing explanation for these seemingly conflicting results is that PLB slows transitions in the SERCA enzymatic cycle associated with Ca2+ binding, altering transport Ca2+ dependence without actually affecting the equilibrium binding affinity of the Ca2+-coordinating sites. Here, we consider another hypothesis, that measurements of Ca2+ binding in the absence of ATP overlook important allosteric effects of nucleotide binding that increase SERCA Ca2+ binding affinity. We speculated that PLB inhibits SERCA by reversing this allostery. To test this, we used a fluorescent SERCA biosensor to quantify the Ca2+ affinity of non-cycling SERCA in the presence and absence of a non-hydrolyzable ATP-analog, AMPPCP. Nucleotide activation increased SERCA Ca2+ affinity, and this effect was reversed by co-expression of PLB. Interestingly, PLB had no effect on Ca2+ affinity in the absence of nucleotide. These results reconcile the previous conflicting observations from ATPase assays versus Ca2+ binding assays. Moreover, structural analysis of SERCA revealed a novel allosteric pathway connecting the ATP- and Ca2+-binding sites. We propose this pathway is disrupted by PLB binding. Thus, PLB reduces the equilibrium Ca2+ affinity of SERCA by interrupting allosteric activation of the pump by ATP.

Genome-wide association study identifies high-impact susceptibility loci for HCC in North America.

BACKGROUND AND AIMS: Despite the substantial impact of environmental factors, individuals with a family history of liver cancer have an increased risk for HCC. However, genetic factors have not been studied systematically by genome-wide approaches in large numbers of individuals from European descent populations (EDP). APPROACH AND RESULTS: We conducted a 2-stage genome-wide association study (GWAS) on HCC not affected by HBV infections. A total of 1872 HCC cases and 2907 controls were included in the discovery stage, and 1200 HCC cases and 1832 controls in the validation. We analyzed the discovery and validation samples separately and then conducted a meta-analysis. All analyses were conducted in the presence and absence of HCV. The liability-scale heritability was 24.4% for overall HCC. Five regions with significant ORs (95% CI) were identified for nonviral HCC: 3p22.1, MOBP , rs9842969, (0.51, [0.40-0.65]); 5p15.33, TERT , rs2242652, (0.70, (0.62-0.79]); 19q13.11, TM6SF2 , rs58542926, (1.49, [1.29-1.72]); 19p13.11 MAU2 , rs58489806, (1.53, (1.33-1.75]); and 22q13.31, PNPLA3 , rs738409, (1.66, [1.51-1.83]). One region was identified for HCV-induced HCC: 6p21.31, human leukocyte antigen DQ beta 1, rs9275224, (0.79, [0.74-0.84]). A combination of homozygous variants of PNPLA3 and TERT showing a 6.5-fold higher risk for nonviral-related HCC compared to individuals lacking these genotypes. This observation suggests that gene-gene interactions may identify individuals at elevated risk for developing HCC. CONCLUSIONS: Our GWAS highlights novel genetic susceptibility of nonviral HCC among European descent populations from North America with substantial heritability. Selected genetic influences were observed for HCV-positive HCC. Our findings indicate the importance of genetic susceptibility to HCC development.

Selective TLR4 Antagonism Prevents and Reverses Morphine-Induced Persistent Postoperative Cognitive Dysfunction, Dysregulation of Synaptic Elements, and Impaired BDNF Signaling in Aged Male Rats.

Perioperative neurocognitive disorders (PNDs) are characterized by confusion, difficulty with executive function, and episodic memory impairment in the hours to months following a surgical procedure. Postoperative cognitive dysfunction (POCD) represents such impairments that last beyond 30 d postsurgery and is associated with increased risk of comorbidities, progression to dementia, and higher mortality. While it is clear that neuroinflammation plays a key role in PND development, what factors underlie shorter self-resolving versus persistent PNDs remains unclear. We have previously shown that postoperative morphine treatment extends POCD from 4 d (without morphine) to at least 8 weeks (with morphine) in aged male rats, and that this effect is likely dependent on the proinflammatory capabilities of morphine via activation of toll-like receptor 4 (TLR4). Here, we extend these findings to show that TLR4 blockade, using the selective TLR4 antagonist lipopolysaccharide from the bacterium Rhodobacter sphaeroides (LPS-RS Ultrapure), ameliorates morphine-induced POCD in aged male rats. Using either a single central preoperative treatment or a 1 week postoperative central treatment regimen, we demonstrate that TLR4 antagonism (1) prevents and reverses the long-term memory impairment associated with surgery and morphine treatment, (2) ameliorates morphine-induced dysregulation of the postsynaptic proteins postsynaptic density 95 and synaptopodin, (3) mitigates reductions in mature BDNF, and (4) prevents decreased activation of the BDNF receptor TrkB (tropomyosin-related kinase B), all at 4 weeks postsurgery. We also reveal that LPS-RS Ultrapure likely exerts its beneficial effects by preventing endogenous danger signal HMGB1 (high-mobility group box 1) from activating TLR4, rather than by blocking continuous activation by morphine or its metabolites. These findings suggest TLR4 as a promising therapeutic target to prevent or treat PNDs.SIGNIFICANCE STATEMENT With humans living longer than ever, it is crucial that we identify mechanisms that contribute to aging-related vulnerability to cognitive impairment. Here, we show that the innate immune receptor toll-like receptor 4 (TLR4) is a key mediator of cognitive dysfunction in aged rodents following surgery and postoperative morphine treatment. Inhibition of TLR4 both prevented and reversed surgery plus morphine-associated memory impairment, dysregulation of synaptic elements, and reduced BDNF signaling. Together, these findings implicate TLR4 in the development of postoperative cognitive dysfunction, providing mechanistic insight and novel therapeutic targets for the treatment of cognitive impairments following immune challenges such as surgery in older individuals.

Quality of Life and Gastrointestinal Symptoms in Long-term Survivors of Pancreatic Cancer Following Pancreatoduodenectomy.

OBJECTIVE: To describe long-term quality of life (QOL) and gastrointestinal (GI) symptoms in patients who underwent pancreatoduodenectomy for pancreatic cancer in the modern era. BACKGROUND: As advances in pancreatic cancer management improve outcomes, it is essential to assess long-term patient-reported outcomes after surgery. METHODS: Patients who underwent curative intent pancreatoduodenectomy for pancreatic cancer between January 2011 and June 2019 from a single center were identified. Patients alive ≥3 years after surgery were considered long-term survivors (LTS). LTS who were alive in June 2022 received a 55-question survey to assess their QOL (EORTC-QLQ-C30) and GI symptoms (EORTC-PAN26 and Problem Areas in Diabetes Questionnaire). Responses were compared against population norms. Clinicodemographic characteristics in LTS versus non-LTS and survey completion were compared. RESULTS: Six hundred seventy-two patients underwent pancreatoduodenectomy for pancreatic cancer; 340 were LTS. One hundred thirty-seven patients of the 238 eligible to complete the survey responded (response rate: 58%). Compared to the US general population, LTS reported significantly higher QOL (75 vs 64; P <0.001), less nausea/vomiting, pain, dyspnea, insomnia, appetite loss, and constipation, but more diarrhea (all P <0.001). Most patients (n=136/137, 99%) reported experiencing postoperative GI symptoms related to pancreatic insufficiency (n=71/135, 53%), reflux (n=61/135, 45%), and delayed gastric emptying (n=31/136, 23%). Most patients (n=113/136, 83%) reported that digestive symptoms overall had little to no impact on QOL, and 91% (n=124/136) would undergo surgery again. CONCLUSIONS: Despite known long-term complications following pancreatoduodenectomy, cancer survivors appear to have excellent QOL. Specific long-term gastrointestinal symptoms data should be utilized for preoperative education and follow-up planning.

Model to predict major complications following liver resection for HCC in patients with metabolic syndrome.

BACKGROUND: Metabolic syndrome (MS) is rapidly growing as risk factor for HCC. Liver resection for HCC in patients with MS is associated with increased postoperative risks. There are no data on factors associated with postoperative complications. AIMS: The aim was to identify risk factors and develop and validate a model for postoperative major morbidity after liver resection for HCC in patients with MS, using a large multicentric Western cohort. MATERIALS AND METHODS: The univariable logistic regression analysis was applied to select predictive factors for 90 days major morbidity. The model was built on the multivariable regression and presented as a nomogram. Performance was evaluated by internal validation through the bootstrap method. The predictive discrimination was assessed through the concordance index. RESULTS: A total of 1087 patients were gathered from 24 centers between 2001 and 2021. Four hundred and eighty-four patients (45.2%) were obese. Most liver resections were performed using an open approach (59.1%), and 743 (68.3%) underwent minor hepatectomies. Three hundred and seventy-six patients (34.6%) developed postoperative complications, with 13.8% major morbidity and 2.9% mortality rates. Seven hundred and thirteen patients had complete data and were included in the prediction model. The model identified obesity, diabetes, ischemic heart disease, portal hypertension, open approach, major hepatectomy, and changes in the nontumoral parenchyma as risk factors for major morbidity. The model demonstrated an AUC of 72.8% (95% CI: 67.2%-78.2%) ( https://childb.shinyapps.io/NomogramMajorMorbidity90days/ ). CONCLUSIONS: Patients undergoing liver resection for HCC and MS are at high risk of postoperative major complications and death. Careful patient selection, considering baseline characteristics, liver function, and type of surgery, is key to achieving optimal outcomes.

Value of Surgical Cytoreduction in Patients with Small Intestinal Neuroendocrine Tumors Metastatic to the Liver and Peritoneum.

BACKGROUND: Cytoreductive hepatectomy can improve survival and symptoms of hormonal excess in patients with small intestinal neuroendocrine tumor (siNET) liver metastases, but whether to proceed when peritoneal metastases are encountered at the time of planned cytoreductive hepatectomy is controversial. METHODS: This was a retrospective review of patients who underwent surgical management of metastatic siNETs at Mayo Clinic between 2000 and 2020. Patients who underwent cytoreductive operation for isolated liver metastases or both liver and peritoneal metastases were compared. RESULTS: Of 261 patients who underwent cytoreductive operation for siNETs, 211 had isolated liver metastases and 50 had liver and peritoneal metastases. Complete cytoreduction was achieved in 78% of patients with isolated liver metastases and 56% of those with liver and peritoneal metastases (p = 0.002). After complete cytoreduction, median overall survival (OS) was 11.5 years for isolated liver metastases and 11.2 years for liver and peritoneal metastases (p = 0.10), and relief of carcinoid syndrome was ≥ 97% in both groups. After incomplete cytoreduction with debulking of > 90% of hepatic disease and/or closing Lyon score of 1-2, median OS was 6.4 years for isolated liver metastases and 7.1 years for liver and peritoneal metastases (p = 0.12). CONCLUSIONS: Patients with siNETs metastatic to both the liver and peritoneum have favorable outcomes after aggressive surgical cytoreduction, with the best outcomes observed after complete cytoreduction. Therefore, the presence of peritoneal metastases should not by itself preclude surgical cytoreduction in this population.

Outcomes of Neoadjuvant Chemotherapy for Invasive Intraductal Papillary Mucinous Neoplasm Compared with de Novo Pancreatic Adenocarcinoma.

BACKGROUND: The management of invasive intraductal papillary mucinous cystic neoplasm (I-IPMN) does not differ from de novo pancreatic ductal adenocarcinoma (PDAC); however, I-IPMNs are debated to have better prognosis. Despite being managed similarly to PDAC, no data are available on the response of I-IPMN to neoadjuvant chemotherapy. METHODS: All patients undergoing pancreatic resection for a pancreatic adenocarcinoma from 2011 to 2022 were included. The PDAC and I-IPMN cohorts were compared to evaluate response to neoadjuvant therapy (NAT) and overall survival (OS). RESULTS: This study included 1052 PDAC patients and 105 I-IPMN patients. NAT was performed in 25% of I-IPMN patients and 65% of PDAC patients. I-IPMN showed a similar pattern of pathological response to NAT compared with PDAC (p = 0.231). Furthermore, positron emission tomography (PET) response (71% vs. 61%; p = 0.447), CA19.9 normalization (85% vs. 76%, p = 0.290), and radiological response (32% vs. 37%, p = 0.628) were comparable between I-IPMN and PDAC. A significantly higher OS and disease-free survival (DFS) of I-IPMN was denoted by Kaplan-Meier analysis, with a p-value of < 0.001 in both plots. In a multivariate analysis, I-IPMN histology was independently associated with lower risk of recurrence and death. CONCLUSIONS: I-IPMN patients have a longer OS and DFS after surgical treatment when compared with PDAC patients. The more favorable oncologic outcome of I-IPMNs does not seem to be related to early detection, as I-IPMN histological subclass is independently associated with a lower risk of disease recurrence. Moreover, neoadjuvant effect on I-IPMN was non-inferior to PDAC in terms of pathological, CA19.9, PET, and radiological response and thus can be considered in selected patients.

Von Willebrand Factor Antigen Improves Risk Stratification for Patients with a Diagnosis of Resectable Hepatocellular Carcinoma.

BACKGROUND: Posthepatectomy liver failure (PHLF), complications of portal hypertension, and disease recurrence determine the outcome for hepatocellular carcinoma (HCC) patients undergoing liver resection. This study aimed to evaluate the von Willebrand factor antigen (vWF-Ag) as a non-invasive test for clinically significant portal hypertension (CSPH) and a predictive biomarker for time to recurrence (TTR) and overall survival (OS). METHODS: The study recruited 72 HCC patients with detailed preoperative workup from a prospective trial (NCT02118545) and followed for complications, TTR, and OS. Additionally, 163 compensated patients with resectable HCC were recruited to evaluate vWF-Ag cutoffs for ruling out or ruling in CSPH. Finally, vWF-Ag cutoffs were prospectively evaluated in an external validation cohort of 34 HCC patients undergoing liver resection. RESULTS: In receiver operating characteristic (ROC) analyses, vWF-Ag (area under the curve [AUC], 0.828) was similarly predictive of PHLF as indocyanine green clearance (disappearance rate: AUC, 0.880; retention rate: AUC, 0.894), whereas computation of future liver remnant was inferior (AUC, 0.756). Cox-regression showed an association of vWF-Ag with TTR (per 10%: hazard ratio [HR], 1.056; 95% confidence interval [CI] 1.017-1.097) and OS (per 10%: HR, 1.067; 95% CI 1.022-1.113). In the analyses, VWF-Ag yielded an AUC of 0.824 for diagnosing CSPH, with a vWF-Ag of 182% or lower ruling out and higher than 291% ruling in CSPH. Therefore, a highest-risk group (> 291%, 9.7% of patients) with a 57.1% incidence of PHLF was identified, whereas no patient with a vWF-Ag of 182% or lower (52.7%) experienced PHLF. The predictive value of vWF-Ag for PHLF and OS was externally validated. CONCLUSION: For patients with resectable HCC, VWF-Ag allows for simplified preoperative risk stratification. Patients with vWF-Ag levels higher than 291% might be considered for alternative treatments, whereas vWF-Ag levels of 182% or lower identify patients best suited for surgery.

Neuroendocrine Tumors of Unknown Primary in the Setting of Cytoreductive Hepatectomy.

BACKGROUND: Surgical cytoreduction for neuroendocrine tumor liver metastasis (NETLM) consistently shows positive long-term outcomes. Despite reservations in guidelines for surgery when the primary tumor is unidentified (UP-NET), this study compared the surgical and oncologic long-term outcomes between patients with these rare cases undergoing cytoreductive surgery and patients who had liver resection for known primaries. METHODS: The study identified 32 unknown primary liver metastases (UP-NETLM) in 522 retrospectively evaluated patients who underwent resection of well-differentiated NETLM between January 2000 and December 2020. Tumor and patient characteristics were compared with those in 490 cases of liver metastasis from small intestinal (SI-NETLM) or pancreatic (pNETLM) primaries. Survival analysis was performed to highlight long-term outcome differences. Surgical outcomes were compared between liver resections alone and simultaneous primary resections to assess surgical risk distinctions. RESULTS: The UP-NET patients had fewer NETLMs (p = 0.004), which on the average were larger than SI-NETLMs or pNETLMs (p = 0.002). Expression of Ki-67 was balanced among the groups. Major hepatectomy was performed more often in the UP-NETLM group (p = 0.017). The 10-year survival rate of 53% for UP-NETLM was comparable with that for SI-NETML (58%; p = 0.463) and pNETLMs (47%; p = 0.497). The median hepatic progression-free survival was 26 months for the UP-NETLM patients and 25 months for the SI-NETLM patients compared to 12 months for the pNETLM patients (p < 0.001). Perioperative mortality was lower than 2%, and severe postoperative morbidity occurred in 21%, similarly distributed among all the groups. CONCLUSION: The surgical risk and long-term outcomes for the UP-NETLM patients were comparable with those for other NETLM cases, affirming the validity of equally aggressive surgical cytoreduction as a therapeutic option in carefully selected cases.

Long-term overall survival in patients undergoing liver resection for metastatic anal squamous cell carcinoma.

BACKGROUND AND OBJECTIVES: There is limited research on management of metastatic anal canal squamous cell carcinoma (SCC) to the liver. This study aimed to describe outcomes for patients undergoing liver resection of anal SCC metastases. METHODS: A multicenter, retrospective cohort study was conducted by three tertiary-referral centers. Patients undergoing liver surgery between 2008 and 2022 were included. Cox regression analysis was performed to evaluate predictors of recurrence and survival and Kaplan-Meier analysis was performed for 1-, 3-, and 5-year survival. RESULTS: Twenty-one patients underwent liver resection and/or ablation. None were HIV positive and 24% had known HPV infection. 20/21(95%) patients had undergone Nigro protocol for management of the primary tumor with 12/21 (57%) patients experiencing complete response. 4/21 (19%) patients had synchronous liver metastases at time of diagnosis. Median tumor size was 5.0 cm and median tumor number was one. At analysis, 52% remained alive. Median overall survival was 32.2 months. 5-year overall survival was 50%. Median recurrence-free survival was 7.7 months and 5-year recurrence-free survival was 30%. Need for salvage abdominoperineal resection was negatively associated with recurrence-free survival. The most common site of recurrence was the liver. CONCLUSIONS: Liver resection for metastatic anal SCC can be beneficial for appropriately selected patients.

Advancing minimally invasive hepato-pancreato-biliary surgery: ensuring safety with implementation.

BACKGROUND: Although minimally invasive hepato-pancreato-biliary (MIS HPB) surgery can be performed with good outcomes, there are currently no standardized requirements for centers or surgeons who wish to implement MIS HPB surgery. The aim of this study was to create a consensus statement regarding safe dissemination and implementation of MIS HPB surgical programs. METHODS: Sixteen key questions regarding safety in MIS HPB surgery were generated after a focused literature search and iterative review by three field experts. Participants for the working group were then selected using sequential purposive sampling and snowball techniques. Review of the 16 questions took place over a single 2-h meeting. The senior author facilitated the session, and a modified nominal group technique was used. RESULTS: Twenty three surgeons were in attendance. All participants agreed or strongly agreed that formal guidelines should exist for both institutions and individual surgeons interested in implementing MIS HPB surgery and that routine monitoring and reporting of institutional and surgeon technical outcomes should be performed. Regarding volume cutoffs, most participants (91%) agreed or strongly agreed that a minimum annual institutional volume cutoff for complex MIS HPB surgery, such as major hepatectomy or pancreaticoduodenectomy, should exist. A smaller proportion (74%) agreed or strongly agreed that a minimum annual surgeon volume requirement should exist. The majority of participants agreed or strongly agreed that surgeons were responsible for defining (100%) and enforcing (78%) guidelines to ensure the overall safety of MIS HPB programs. Finally, formal MIS HPB training, minimum case volume requirements, institutional support and infrastructure, and mandatory collection of outcomes data were all recognized as important aspects of safe implementation of MIS HPB surgery. CONCLUSIONS: Safe implementation of MIS HPB surgery requires a thoughtful process that incorporates structured training, sufficient volume and expertise, a proper institutional ecosystem, and monitoring of outcomes.

Supply and demand of hepatopancreatobiliary surgeons in the United States.

BACKGROUND: Hepatopancreatobiliary (HPB) surgery requires specialized training and adequate case volumes to maintain procedural proficiency and optimal outcomes. Studies of individual HPB surgeon supply related to annual HPB case demand are sparse. This study assesses the supply and demand of the HPB surgical workforce in the United States (US). METHODS: The National Inpatient Sample (NIS) was queried from 1998-2019 to estimate the number of HPB procedures performed. To approximate the number of HPB surgeons, models based on previous HPB workforce publications were employed. We then calculated the number of HPB surgeons needed to maintain volume-outcome thresholds at current reported levels of centralization. RESULTS: In 2019, approximately 37,335 patients underwent inpatient HPB procedures in the US, while an estimated 905-1191 HPB surgeons were practicing. Assuming 50% centralization and an optimal volume-outcome threshold of 24 HPB cases-per-year, only 778 HPB surgeons were needed. Without adjustment in centralization, by 2030 there will be a demand of fewer than 12 annual cases per HPB surgeon. CONCLUSION: The current supply of HPB surgeons may exceed demand in the United States. Without alteration in training pathways or improved care centralization, by 2030, there will be insufficient HPB case volume per surgeon to maintain published volume-outcome standards.

Early vs conventional onset pancreatic ductal adenocarcinoma: analysis of surgical and oncologic outcomes in patients undergoing curative intent resection.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) impacts patients in their 60s, but its incidence in younger patients is increasing. We hypothesize that younger patients may have worse oncologic outcomes. METHODS: Patients who underwent curative pancreatic resection for PDAC between January 2011 and December 2021 at a single institution were analyzed. Early-onset pancreatic cancer (EOPC) was defined as pancreatic cancer diagnosed in patients ≤50 years. Clinical and survival outcomes were compared between EOPC and Conventional Onset Pancreas Cancer (COPC). RESULTS: A total of 1133 patients were identified, 65 (5.7%) were EOPC. Preoperative patient characteristics including sex, smoking status, alcohol habitus, diabetes mellitus, CA 19-9, and neoadjuvant therapy were similar between EOPC and COPC (p > 0.05). EOPC patients were more likely non-white (p = 0.03), had lower ASA scores (p = 0.02) and larger median tumor size (33 vs 28 mm, p = 0.04), but had similar pathological stages and rate of R0 resections (p > 0.05). Postoperative outcomes were similar (p > 0.05). There was no statistically significant difference in overall (HR 0.93, CI 0.64, 1.33; p = 0.68) or recurrence free (HR 1.05, CI 0.75, 1.48; p = 0.77) survival between the EOPC and COPC after adjusting for significant factors. CONCLUSION: Patients with EOPC who underwent surgical resection had similar oncological outcomes compared to patients with COPC.

Micropeptide hetero-oligomerization adds complexity to the calcium pump regulatory network.

The sarco(endo)plasmic reticulum calcium ATPase (SERCA) is an ion transporter that creates and maintains intracellular calcium stores. SERCA is inhibited or stimulated by several membrane micropeptides including another-regulin, dwarf open reading frame, endoregulin, phospholamban (PLB), and sarcolipin. We previously showed that these micropeptides assemble into homo-oligomeric complexes with varying affinity. Here, we tested whether different micropeptides can interact with each other, hypothesizing that coassembly into hetero-oligomers may affect micropeptide bioavailability to regulate SERCA. We quantified the relative binding affinity of each combination of candidates using automated fluorescence resonance energy transfer microscopy. All pairs were capable of interacting with good affinity, similar to the affinity of micropeptide self-binding (homo-oligomerization). Testing each pair at a 1:5 ratio and a reciprocal 5:1 ratio, we noted that the affinity of hetero-oligomerization of some micropeptides depended on whether they were the minority or majority species. In particular, sarcolipin was able to join oligomers when it was the minority species but did not readily accommodate other micropeptides in the reciprocal experiment when it was expressed in fivefold excess. The opposite was observed for endoregulin. PLB was a universal partner for all other micropeptides tested, forming avid hetero-oligomers whether it was the minority or majority species. Increasing expression of SERCA decreased PLB-dwarf open reading frame hetero-oligomerization, suggesting that SERCA-micropeptide interactions compete with micropeptide-micropeptide interactions. Thus, micropeptides populate a regulatory network of diverse protein assemblies. The data suggest that the complexity of this interactome increases exponentially with the number of micropeptides that are coexpressed in a particular tissue.

Inhibitory and stimulatory micropeptides preferentially bind to different conformations of the cardiac calcium pump.

The ATP-dependent ion pump sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) sequesters Ca2+ in the endoplasmic reticulum to establish a reservoir for cell signaling. Because of its central importance in physiology, the activity of this transporter is tightly controlled via direct interactions with tissue-specific regulatory micropeptides that tune SERCA function to match changing physiological conditions. In the heart, the micropeptide phospholamban (PLB) inhibits SERCA, while dwarf open reading frame (DWORF) stimulates SERCA. These competing interactions determine cardiac performance by modulating the amplitude of Ca2+ signals that drive the contraction/relaxation cycle. We hypothesized that the functions of these peptides may relate to their reciprocal preferences for SERCA binding; SERCA binds PLB more avidly at low cytoplasmic [Ca2+] but binds DWORF better when [Ca2+] is high. In the present study, we demonstrated this opposing Ca2+ sensitivity is due to preferential binding of DWORF and PLB to different intermediate states that SERCA samples during the Ca2+ transport cycle. We show PLB binds best to the SERCA E1-ATP state, which prevails at low [Ca2+]. In contrast, DWORF binds most avidly to E1P and E2P states that are more populated when Ca2+ is elevated. Moreover, FRET microscopy revealed dynamic shifts in SERCA-micropeptide binding equilibria during cellular Ca2+ elevations. A computational model showed that DWORF exaggerates changes in PLB-SERCA binding during the cardiac cycle. These results suggest a mechanistic basis for inhibitory versus stimulatory micropeptide function, as well as a new role for DWORF as a modulator of dynamic oscillations of PLB-SERCA regulatory interactions.

Fluorescence lifetime imaging microscopy reveals sodium pump dimers in live cells.

The sodium-potassium ATPase (Na/K-ATPase, NKA) establishes ion gradients that facilitate many physiological functions including action potentials and secondary transport processes. NKA comprises a catalytic subunit (alpha) that interacts closely with an essential subunit (beta) and regulatory transmembrane micropeptides called FXYD proteins. In the heart, a key modulatory partner is the FXYD protein phospholemman (PLM, FXYD1), but the stoichiometry of the alpha-beta-PLM regulatory complex is unknown. Here, we used fluorescence lifetime imaging and spectroscopy to investigate the structure, stoichiometry, and affinity of the NKA-regulatory complex. We observed a concentration-dependent binding of the subunits of NKA-PLM regulatory complex, with avid association of the alpha subunit with the essential beta subunit as well as lower affinity alpha-alpha and alpha-PLM interactions. These data provide the first evidence that, in intact live cells, the regulatory complex is composed of two alpha subunits associated with two beta subunits, decorated with two PLM regulatory subunits. Docking and molecular dynamics (MD) simulations generated a structural model of the complex that is consistent with our experimental observations. We propose that alpha-alpha subunit interactions support conformational coupling of the catalytic subunits, which may enhance NKA turnover rate. These observations provide insight into the pathophysiology of heart failure, wherein low NKA expression may be insufficient to support formation of the complete regulatory complex with the stoichiometry (alpha-beta-PLM)2.

An APRI+ALBI Based Multivariable Model as Preoperative Predictor for Posthepatectomy Liver Failure.

OBJECTIVE AND BACKGROUND: Clinically significant posthepatectomy liver failure (PHLF B+C) remains the main cause of mortality after major hepatic resection. This study aimed to establish an APRI+ALBI, aspartate aminotransferase to platelet ratio (APRI) combined with albumin-bilirubin grade (ALBI), based multivariable model (MVM) to predict PHLF and compare its performance to indocyanine green clearance (ICG-R15 or ICG-PDR) and albumin-ICG evaluation (ALICE). METHODS: 12,056 patients from the National Surgical Quality Improvement Program (NSQIP) database were used to generate a MVM to predict PHLF B+C. The model was determined using stepwise backwards elimination. Performance of the model was tested using receiver operating characteristic curve analysis and validated in an international cohort of 2,525 patients. In 620 patients, the APRI+ALBI MVM, trained in the NSQIP cohort, was compared with MVM's based on other liver function tests (ICG clearance, ALICE) by comparing the areas under the curve (AUC). RESULTS: A MVM including APRI+ALBI, age, sex, tumor type and extent of resection was found to predict PHLF B+C with an AUC of 0.77, with comparable performance in the validation cohort (AUC 0.74). In direct comparison with other MVM's based on more expensive and time-consuming liver function tests (ICG clearance, ALICE), the APRI+ALBI MVM demonstrated equal predictive potential for PHLF B+C. A smartphone application for calculation of the APRI+ALBI MVM was designed. CONCLUSION: Risk assessment via the APRI+ALBI MVM for PHLF B+C increases preoperative predictive accuracy and represents an universally available and cost-effective risk assessment prior to hepatectomy, facilitated by a freely available smartphone app.

Minimally Invasive Versus Open Liver Resections for Hepatocellular Carcinoma in Patients With Metabolic Syndrome.

OBJECTIVE: To compare minimally invasive (MILR) and open liver resections (OLRs) for hepatocellular carcinoma (HCC) in patients with metabolic syndrome (MS). BACKGROUND: Liver resections for HCC on MS are associated with high perioperative morbidity and mortality. No data on the minimally invasive approach in this setting exist. MATERIAL AND METHODS: A multicenter study involving 24 institutions was conducted. Propensity scores were calculated, and inverse probability weighting was used to weight comparisons. Short-term and long-term outcomes were investigated. RESULTS: A total of 996 patients were included: 580 in OLR and 416 in MILR. After weighing, groups were well matched. Blood loss was similar between groups (OLR 275.9±3.1 vs MILR 226±4.0, P =0.146). There were no significant differences in 90-day morbidity (38.9% vs 31.9% OLRs and MILRs, P =0.08) and mortality (2.4% vs 2.2% OLRs and MILRs, P =0.84). MILRs were associated with lower rates of major complications (9.3% vs 15.3%, P =0.015), posthepatectomy liver failure (0.6% vs 4.3%, P =0.008), and bile leaks (2.2% vs 6.4%, P =0.003); ascites was significantly lower at postoperative day 1 (2.7% vs 8.1%, P =0.002) and day 3 (3.1% vs 11.4%, P <0.001); hospital stay was significantly shorter (5.8±1.9 vs 7.5±1.7, P <0.001). There was no significant difference in overall survival and disease-free survival. CONCLUSIONS: MILR for HCC on MS is associated with equivalent perioperative and oncological outcomes to OLRs. Fewer major complications, posthepatectomy liver failures, ascites, and bile leaks can be obtained, with a shorter hospital stay. The combination of lower short-term severe morbidity and equivalent oncologic outcomes favor MILR for MS when feasible.

Combining Surface-Induced Dissociation and Charge Detection Mass Spectrometry to Reveal the Native Topology of Heterogeneous Protein Complexes.

Charge detection mass spectrometry (CDMS) enables the direct mass measurement of heterogeneous samples on the megadalton scale, as the charge state for a single ion is determined simultaneously with the mass-to-charge ratio (m/z). Surface-induced dissociation (SID) is an effective activation method to dissociate non-intertwined, non-covalent protein complexes without extensive gas-phase restructuring, producing various subcomplexes reflective of the native protein topology. Here, we demonstrate that using CDMS after SID on an Orbitrap platform offers subunit connectivity, topology, proteoform information, and relative interfacial strengths of the intact macromolecular assemblies. SID dissects the capsids (∼3.7 MDa) of adeno-associated viruses (AAVs) into trimer-containing fragments (3mer, 6mer, 9mer, 15mer, etc.) that can be detected by the individual ion mass spectrometry (I2MS) approach on Orbitrap instruments. SID coupled to CDMS provides unique structural insights into heterogeneous assemblies that are not readily obtained by traditional MS measurements.

Dynamic risk profiling of HCC recurrence after curative intent liver resection.

BACKGROUND AND AIMS: Following liver resection (LR) for HCC, the likelihood of survival is dynamic, in that multiple recurrences and/or metastases are possible, each having variable impacts on outcomes. We sought to evaluate the natural progression, pattern, and timing of various disease states after LR for HCC using multistate modeling and to create a practical calculator to provide prognostic information for patients and clinicians. APPROACH AND RESULTS: Adult patients undergoing LR for HCC between January 2000 and December 2018 were retrospectively identified at a single center. Multistate analysis modeled post-LR tumor progression by describing transitions between distinct disease states. In this model, the states included surgery, intrahepatic recurrence (first, second, third, fourth, fifth), distant metastasis with or without intrahepatic recurrence, and death. Of the 486 patients included, 169 (34.8%) remained recurrence-free, 205 (42.2%) developed intrahepatic recurrence, 80 (16.5%) developed distant metastasis, and 32 (7%) died. For an average patient having undergone LR, there was a 33.1% chance of remaining disease-free, a 31.0% chance of at least one intrahepatic recurrence, a 16.3% chance of distant metastasis, and a 19.8% chance of death within the first 60 months post-LR. The transition probability from surgery to first intrahepatic recurrence, without a subsequent state transition, increased from 3% (3 months) to 17.4% (30 months) and 17.2% (60 months). Factors that could modify these probabilities included tumor size, satellite lesions, and microvascular invasion. The online multistate model calculator can be found on https://multistatehcc.shinyapps.io/home/. CONCLUSIONS: In contrast to standard single time-to-event estimates, multistate modeling provides more realistic prognostication of outcomes after LR for HCC by taking into account many postoperative disease states and transitions between them. Our multistate modeling calculator can provide meaningful data to guide the management of patients undergoing postoperative surveillance and therapy.