Engineering cellular communication between light-activated synthetic cells and bacteria.

Gene-expressing compartments assembled from simple, modular parts, are a versatile platform for creating minimal synthetic cells with life-like functions. By incorporating gene regulatory motifs into their encapsulated DNA templates, in situ gene expression and, thereby, synthetic cell function can be controlled according to specific stimuli. In this work, cell-free protein synthesis within synthetic cells was controlled using light by encoding genes of interest on light-activated DNA templates. Light-activated DNA contained a photocleavable blockade within the T7 promoter region that tightly repressed transcription until the blocking groups were removed with ultraviolet light. In this way, synthetic cells were activated remotely, in a spatiotemporally controlled manner. By applying this strategy to the expression of an acyl homoserine lactone synthase, BjaI, quorum-sensing-based communication between synthetic cells and bacteria was controlled with light. This work provides a framework for the remote-controlled production and delivery of small molecules from nonliving matter to living matter, with applications in biology and medicine.

Precise, Orthogonal Remote-Control of Cell-Free Systems Using Photocaged Nucleic Acids.

Cell-free expression of a gene to protein has become a vital tool in nanotechnology and synthetic biology. Remote-control of cell-free systems with multiple, orthogonal wavelengths of light would enable precise, noninvasive modulation, opening many new applications in biology and medicine. While there has been success in developing ON switches, the development of OFF switches has been lacking. Here, we have developed orthogonally light-controlled cell-free expression OFF switches by attaching nitrobenzyl and coumarin photocages to antisense oligonucleotides. These light-controlled OFF switches can be made from commercially available oligonucleotides and show a tight control of cell-free expression. Using this technology, we have demonstrated orthogonal degradation of two different mRNAs, depending on the wavelength used. By combining with our previously generated blue-light-activated DNA template ON switch, we were able to start transcription with one wavelength of light and then halt the translation of the corresponding mRNA to protein with a different wavelength, at multiple timepoints. This precise, orthogonal ON and OFF remote-control of cell-free expression will be an important tool for the future of cell-free biology, especially for use with biological logic gates and synthetic cells.

Orthogonal Light-Activated DNA for Patterned Biocomputing within Synthetic Cells.

Cell-free gene expression is a vital research tool to study biological systems in defined minimal environments and has promising applications in biotechnology. Developing methods to control DNA templates for cell-free expression will be important for precise regulation of complex biological pathways and use with synthetic cells, particularly using remote, nondamaging stimuli such as visible light. Here, we have synthesized blue light-activatable DNA parts that tightly regulate cell-free RNA and protein synthesis. We found that this blue light-activated DNA could initiate expression orthogonally to our previously generated ultraviolet (UV) light-activated DNA, which we used to generate a dual-wavelength light-controlled cell-free AND-gate. By encapsulating these orthogonal light-activated DNAs into synthetic cells, we used two overlapping patterns of blue and UV light to provide precise spatiotemporal control over the logic gate. Our blue and UV orthogonal light-activated DNAs will open the door for precise control of cell-free systems in biology and medicine.

Reaction-Diffusion Patterning of DNA-Based Artificial Cells.

Biological cells display complex internal architectures with distinct micro environments that establish the chemical heterogeneity needed to sustain cellular functions. The continued efforts to create advanced cell mimics, namely, artificial cells, demands strategies for constructing similarly heterogeneous structures with localized functionalities. Here, we introduce a platform for constructing membraneless artificial cells from the self-assembly of synthetic DNA nanostructures in which internal domains can be established thanks to prescribed reaction-diffusion waves. The method, rationalized through numerical modeling, enables the formation of up to five distinct concentric environments in which functional moieties can be localized. As a proof-of-concept, we apply this platform to build DNA-based artificial cells in which a prototypical nucleus synthesizes fluorescent RNA aptamers that then accumulate in a surrounding storage shell, thus demonstrating the spatial segregation of functionalities reminiscent of that observed in biological cells.

Re-interventions and re-admissions in a 13-year series following use of laparoscopic subtotal cholecystectomy.

BACKGROUND: Laparoscopic subtotal cholecystectomy (LSTC) without cystic duct ligation is an alternative to conversion to open surgery in a difficult cholecystectomy, thus avoiding a potentially hazardous dissection in Calot's triangle. The long-term outcomes of this procedure are not well reported. The aim of this study is to assess the rates of re-presentation, re-admissions, endoscopic interventions and completion cholecystectomy in patients who have undergone LSTC. METHODS: Details of all patients undergoing cholecystectomy over a 13-year period (2003-2015) were entered on a prospective database. Further information on subsequent hospital attendances, biliary imaging, endoscopic interventions and re-operations following the index LSTC was collected retrospectively from hospital database. RESULTS: Overall, 2313 patients underwent laparoscopic cholecystectomy. Eighty-five patients (3.7%) underwent LSTC and the rest had standard laparoscopic cholecystectomy. A controlled bile leak was observed in 16 (19%) patients post-operatively, of which 3 resolved spontaneously. The remaining 13 were managed with an early endoscopic retrograde cholangiopancreatography (ERCP) and biliary stent. Twenty-seven patients (32%), who underwent LSTC, were re-investigated for the upper abdominal symptoms. The time range for re-investigation was 21 days-124 months. Eight patients underwent ERCP post-discharge, for suspected bile duct stones on radiological imaging. Two patients required open completion cholecystectomy for symptomatic stones in the gallbladder remnant. CONCLUSION: LSTC is a feasible and safe alternative to open surgery with acceptable long-term consequences and re-interventions.

Controlling gene expression with light: a multidisciplinary endeavour.

The expression of a gene to a protein is one of the most vital biological processes. The use of light to control biology offers unparalleled spatiotemporal resolution from an external, orthogonal signal. A variety of methods have been developed that use light to control the steps of transcription and translation of specific genes into proteins, for cell-free to in vivo biotechnology applications. These methods employ techniques ranging from the modification of small molecules, nucleic acids and proteins with photocages, to the engineering of proteins involved in gene expression using naturally light-sensitive proteins. Although the majority of currently available technologies employ ultraviolet light, there has been a recent increase in the use of functionalities that work at longer wavelengths of light, to minimise cellular damage and increase tissue penetration. Here, we discuss the different chemical and biological methods employed to control gene expression, while also highlighting the central themes and the most exciting applications within this diverse field.

British Society of Gastroenterology guidelines for oesophageal manometry and oesophageal reflux monitoring.

These guidelines on oesophageal manometry and gastro-oesophageal reflux monitoring supersede those produced in 2006. Since 2006 there have been significant technological advances, in particular, the development of high resolution manometry (HRM) and oesophageal impedance monitoring. The guidelines were developed by a guideline development group of patients and representatives of all the relevant professional groups using the Appraisal of Guidelines for Research and Evaluation (AGREE II) tool. A systematic literature search was performed and the GRADE (Grading of Recommendations Assessment, Development and Evaluation) tool was used to evaluate the quality of evidence and decide on the strength of the recommendations made. Key strong recommendations are made regarding the benefit of: (i) HRM over standard manometry in the investigation of dysphagia and, in particular, in characterising achalasia, (ii) adjunctive testing with larger volumes of water or solids during HRM, (iii) oesophageal manometry prior to antireflux surgery, (iv) pH/impedance monitoring in patients with reflux symptoms not responding to high dose proton pump inhibitors and (v) pH monitoring in all patients with reflux symptoms responsive to proton pump inhibitors in whom surgery is planned, but combined pH/impedance monitoring in those not responsive to proton pump inhibitors in whom surgery is planned. This work has been endorsed by the Clinical Services and Standards Committee of the British Society of Gastroenterology (BSG) under the auspices of the oesophageal section of the BSG.

Fibroblast growth factor 23 levels decline following sleeve gastrectomy.

OBJECTIVE: Levels of fibroblast growth factor 23 (FGF23) have been positively associated with measures of adiposity, cardiovascular disease and mortality. It is unclear whether the relationship of FGF23 with cardiovascular disease and mortality is confounded by obesity. We aimed to determine whether FGF23 concentrations decline following a reduction in adiposity after sleeve gastrectomy (SG). DESIGN: The effect of SG on FGF23 was evaluated in 22 obese adults (59% male) with type 2 diabetes. Fat mass, weight, BMI, plasma intact FGF23, parathyroid hormone (PTH) and leptin were determined at baseline and at 12 months following SG. RESULTS: At baseline, median (IQR) age was 51 (43-54) years, fat mass 47.8 (41.0-59.4) kg, BMI 40.9 (36.9-46.9) kg/m2 and FGF23 66.2 (55.3-82.9) pg/mL. Significant changes in median BMI (-10.8 kg/m2 , 95% CI: -12.9 to -7.2, P < 0.0001), fat mass (-20.0 kg, 95% CI: -26.7 to -12.4, P < 0.0001) and weight (-34.7 kg, 95% CI -40.0 to -23.1, P < 0.0001) were observed after SG. FGF23 (-12.4 pg/mL, 95% CI: -19.5 to 2.0, P = 0.005), PTH (-1.1 pmol/L, 95% CI: -1.7 to 0.2, P = 0.009) and leptin (-1687 pg/mL, 95% CI -4524 to -563, P = 0.01) declined following SG. Change in FGF23 was not significantly associated with change in measures of adiposity, PTH or leptin. CONCLUSIONS: FGF23 concentrations decline in the setting of significant weight loss following SG, implying that increased FGF23 concentrations are a downstream consequence of obesity, which may confound its association with cardiometabolic dysfunction. Mediators of the relationship between adiposity and FGF23 require further elucidation.

Variation in public-funded bariatric surgery intervention rate by New Zealand region.

Inequitable access to bariatric surgery by geographical region has been reported internationally, but comparable data on provision of bariatric surgery have not previously been reported in New Zealand. We examined allocated funding and provision of bariatric surgery amongst different regions in New Zealand in the 2013/14 year, and found that there was large variation in both. This highlights that public funded bariatric surgery needs to take into account population prevalence of morbid obesity to reduce inequities by geographical region.

Diagnostic inflammatory markers in acute cholangitis.

BACKGROUND: The 2018 Tokyo guidelines for acute cholangitis (AC) use white cell count (WCC) as one of the diagnostic criteria. However, the 2018 Tokyo guidelines grading does not provide guidance for AC patients with normal WCC. In this situation, other inflammatory biomarkers also can be used to diagnose AC and grade severity, but their diagnostic values are yet undetermined. The aims of this study were to evaluate the discriminative powers of common inflammatory markers compared with WCC for diagnosing AC and to determine their diagnostic cutoff levels. METHODS: This was a retrospective cohort study. Over 2 y, 96 patients who underwent endoscopic biliary decompression were identified from the Auckland City Hospital Radiology Department database. Only patients with a confirmed diagnosis of AC were included in the study. Thirty-four patients with AC and 18 controls met eligibility criteria. RESULTS: Comparing areas under the receiver operating characteristic curves, it was the lymphocyte count, neutrophil-to-lymphocyte ratio (NLR), and C-reactive protein (CRP) that had the highest discriminative powers in diagnosing AC. Values of WCC for diagnosing AC were equal to or above 9.6 × 109/L, neutrophil count equal to or exceeding 4.9 × 109/L, lymphocyte count equal to or below 1.3 × 109/L, NLR 5.3 and above, albumin equal to or below 30.5 g/L, and CRP concentration 23.5 mg/L or above. CONCLUSIONS: Lymphocyte count, NLR, and CRP have superior discriminative powers to WCC, albumin, and neutrophil count and can be useful in the diagnosis of AC.

Evaluation of neutrophil-to-lymphocyte ratio as a potential biomarker for acute cholecystitis.

BACKGROUND: The diagnosis of acute cholecystitis (AC) is frequently associated with an increase in white cell count (WCC) and C-reactive protein (CRP). However, one or both of these inflammatory biomarkers can be normal in AC. The aim of this study was to evaluate and compare the discriminative powers of the neutrophil-to-lymphocyte ratio (NLR) with WCC and CRP in diagnosing AC. METHODS: This was a retrospective cohort study. For more than a period of 5 y, 1959 patients were identified from the cholecystectomy Registry. Laparoscopic cholecystectomy patients with histologic evidence of AC were included if they also had preoperative WCC and CRP measurements. Eligibility criteria were met by 177 patients. These patients were compared with 45 control subjects who had normal gallbladder histology. RESULTS: One unit of increase in the NLR was associated with a 2.5 times increase in the odds of AC (odds ratio = 2.48; 95% confidence interval [CI], 1.5-4.1; P < 0.0005). NLR cutoff values of 4.1 (95% CI, 3.42-4.79), 3.25 (95% CI, 1.95-4.54), and 4.17 (95% CI, 3.76-4.58) were diagnostic for the overall AC, mild, and moderate-severe AC, respectively. The NLR areas under the receiver operating characteristic curve in AC, mild, and moderate-severe AC were 94% (95% CI, 91%-97%), 87% (95% CI, 81%-93%), and 98% (95% CI, 96%-100%), respectively. The discriminative power of an NLR was superior to that of the WCC and similar to CRP for diagnosing AC and different grades of severity. CONCLUSIONS: NLR can be considered as a potential inflammatory biomarker for AC.

Costly Cures.

Effective but very expensive drugs are forcing state Medicaid directors to make some tough decisions.

Dissection of Cdk1-cyclin complexes in vivo.

Cyclin-dependent kinases (Cdks) are regulatory enzymes with temporal and spatial selectivity for their protein substrates that are governed by cell cycle-regulated cyclin subunits. Specific cyclin-Cdk complexes bind to and phosphorylate target proteins, coupling their activity to cell cycle states. The identification of specific cyclin-Cdk substrates is challenging and so far, has largely been achieved through indirect correlation or use of in vitro techniques. Here, we use a protein-fragment complementation assay based on the optimized yeast cytosine deaminase to systematically identify candidate substrates of budding yeast Saccharomyces cerevisiae Cdk1 and show dependency on one or more regulatory cyclins. We identified known and candidate cyclin dependencies for many predicted protein kinase Cdk1 targets and showed elusory Clb3-Cdk1-specific phosphorylation of γ-tubulin, thus establishing the timing of this event in controlling assembly of the mitotic spindle. Our strategy can be generally applied to identify substrates and accessory subunits of multisubunit protein complexes.

C-reactive protein measurement is not associated with an improved management of acute cholecystitis: a plié for a change.

BACKGROUND: The diagnosis of acute cholecystitis (AC) is challenging and may result in a delay in surgical treatment and increased mortality. The 2007 and 2013 Tokyo Guidelines for AC proposed to use C-reactive protein (CRP) as an additional bench mark of AC. The aim of this study was to evaluate whether CRP measurement influences management of patients with AC. METHODS: For more than a period of 5 y (May 2004 to June 2009), 1959 patients were identified from the audit of cholecystectomies in North Shore, Waitakere and Southern Cross hospitals at Waitemata District Health Board, Auckland, New Zealand. The exclusion criteria were elective and private patients, patients without AC on histologic examination of gallbladders, and patients with acute acalculous cholecystitis. RESULTS: A total of 414 patients met eligibility criteria. Compared with the non-CRP group, patients who had CRP measured had a longer time to operation theater and a greater proportion of acute gangrenous cholecystitis on histologic examination of excised gallbladders, but similar postoperative complication rate, index, and total hospital stay. Time to operation theater was not associated with development of acute gangrenous cholecystitis (odds ratio, 1.0; 95% confidence interval, 0.996-1.01; P = 0.797), but correlated with the index hospital admission length (correlation coefficient, 0.6092; P < 0.001). CONCLUSIONS: CRP measurement does not influence management of patients with AC. To improve quality of care and to minimize health care provider costs physiologically fit patients with more advanced forms of AC and higher values of CRP should have their operation performed earlier than patients with mild AC and a lower concentration of CRP.

C-reactive protein has a better discriminative power than white cell count in the diagnosis of acute cholecystitis.

BACKGROUND: The diagnosis of acute cholecystitis (AC) is challenging and may result in a delay in surgery, hospital discharge, and increased mortality. To improve its diagnosis, C-reactive protein (CRP) has been proposed as a benchmark. The aim of this study was to evaluate discriminative power of CRP against white cell count (WCC) in AC. METHODS: This was a retrospective cohort study. Over a 5-y period, 1959 patients were identified from the audit of cholecystectomies. The exclusion criteria were coexisting acute surgical conditions, absence of blood tests within 3 d before hospital admission for elective surgery, and private patients. RESULTS: The eligibility criteria were met by 1843 patients. Comparison of the area under receiver operating characteristic (AUC) curve of CRP and WCC in acute on chronic, edematous, necrotic, suppurative, and gangrenous AC showed a better discriminative power of CRP. Both tests performed equally well in patients with pericholecystic abscess and gallbladder perforation. CRP was superior than WCC in mild AC, AUC = 0.93 (95% confidence interval [CI], 0.9-0.95) and 0.79 (95% CI, 0.74-0.84), P < 0.00005, in moderate and severe AC, AUC = 0.99 (95% CI, 0.97-1.0) and 0.92 (95% CI, 0.88-0.97), P = 0.009, and in all forms of AC combined, AUC = 0.94; (95% CI, 0.92-0.97) and 0.83 (95% CI, 0.79-0.87), respectively, P < 0.00005. CONCLUSIONS: CRP has a better discriminative power than WCC in most forms of AC and is a useful diagnostic marker of AC.

Long-term safety and effectiveness of mechanical versus biologic aortic valve prostheses in older patients: results from the Society of Thoracic Surgeons Adult Cardiac Surgery National Database.

BACKGROUND: There is a paucity of long-term data comparing biological versus mechanical aortic valve prostheses in older individuals. METHODS AND RESULTS: We performed follow-up of patients aged 65 to 80 years undergoing aortic valve replacement with a biological (n=24 410) or mechanical (n=14 789) prosthesis from 1991 to 1999 at 605 centers within the Society of Thoracic Surgeons Adult Cardiac Surgery Database using Medicare inpatient claims (mean, 12.6 years; maximum, 17 years; minimum, 8 years), and outcomes were compared by propensity methods. Among Medicare-linked patients undergoing aortic valve replacement (mean age, 73 years), both reoperation (4.0%) and endocarditis (1.9%) were uncommon to 12 years; however, the risk for other adverse outcomes was high, including death (66.5%), stroke (14.1%), and bleeding (17.9%). Compared with those receiving a mechanical valve, patients given a bioprosthesis had a similar adjusted risk for death (hazard ratio, 1.04; 95% confidence interval, 1.01-1.07), higher risks for reoperation (hazard ratio, 2.55; 95% confidence interval, 2.14-3.03) and endocarditis (hazard ratio, 1.60; 95% confidence interval, 1.31-1.94), and lower risks for stroke (hazard ratio, 0.87; 95% confidence interval, 0.82-0.93) and bleeding (hazard ratio, 0.66; 95% confidence interval, 0.62-0.70). Although these results were generally consistent among patient subgroups, bioprosthesis patients aged 65 to 69 years had a substantially elevated 12-year absolute risk of reoperation (10.5%). CONCLUSIONS: Among patients undergoing aortic valve replacement, long-term mortality rates were similar for those who received bioprosthetic versus mechanical valves. Bioprostheses were associated with a higher long-term risk of reoperation and endocarditis but a lower risk of stroke and hemorrhage. These risks varied as a function of a patient's age and comorbidities.

Effect of race and ethnicity on outcomes with drug-eluting and bare metal stents: results in 423 965 patients in the linked National Cardiovascular Data Registry and centers for Medicare & Medicaid services payer databases.

BACKGROUND: Black, Hispanic, and Asian patients have been underrepresented in percutaneous coronary intervention clinical trials; therefore, there are limited data available on outcomes for these race/ethnicity groups. METHODS AND RESULTS: We examined outcomes in 423 965 patients in the National Cardiovascular Data Registry CathPCI Registry database linked to Medicare claims for follow-up. Within each race/ethnicity group, we examined trends in drug-eluting stent (DES) use, 30-month outcomes, and relative outcomes of DES versus bare metal stents. Overall, 390 351 white, 20 191 black, 9342 Hispanic, and 4171 Asian patients > 65 years of age underwent stent implantation from 2004 through 2008 at 940 National Cardiovascular Data Registry participating sites. Trends in adoption of DES were similar across all groups. Relative to whites, black and Hispanic patients undergoing percutaneous coronary intervention had higher long-term risks of death and myocardial infarction (blacks: hazard ratio, 1.28; 95% confidence interval, 1.24-1.32; Hispanics: hazard ratio, 1.15; 95% confidence interval, 1.10-1.21). Long-term outcomes were similar in Asians and whites (hazard ratio, 0.99; 95% confidence interval, 0.92-1.08). Use of DES was associated with better 30-month survival and lower myocardial infarction rates compared with the use of bare metal stents among all race/ethnicity groups except Hispanics, who had similar outcomes with DES or bare metal stents. CONCLUSIONS: Black and Hispanic patients undergoing percutaneous coronary intervention had worse long-term outcomes relative to white and Asian patients. Compared with bare metal stent use, DES use was generally associated with superior long-term outcomes in all racial and ethnic groups, although these differences were not statistically significant in Hispanic patients.

Comparative effectiveness of drug-eluting stents on long-term outcomes in elderly patients treated for in-stent restenosis: a report from the National Cardiovascular Data Registry.

OBJECTIVE: We assessed the long-term outcomes of elderly patients who had in-stent restenosis (ISR) treated with drug-eluting stents (DES) compared with other treatment strategies. BACKGROUND: Elderly patients with ISR represent a vulnerable group of which little is known regarding the safety and efficacy of repeat percutaneous coronary intervention (PCI). METHODS: We analyzed patients ≥ 65 years of age who underwent PCI for ISR in the National Cardiovascular Data Registry(®) from 2004 to 2008. Death, myocardial infarction (MI), revascularization, stroke, and bleeding were assessed for up to 30 months by a linkage with Medicare rehospitalization claims. RESULTS: Of 43,679 linked patients, 30,012 were treated with DES, 8,277 with balloon angioplasty (BA), and 4,917 with bare metal stents (BMS). Compared with BMS, DES use was associated with a lower propensity score-matched (PM) risk of death (hazard ratio [HR] 0.72; 95% confidence interval [CI] 0.66-0.80, P < 0.001), MI (HR 0.81; 95% CI 0.70-0.93, P = 0.003), and revascularization (HR 0.90; 95% CI 0.82-1.00, P = 0.055). Compared with BA, DES use was associated with a lower PM risk of death (HR 0.82; 95% CI 0.76-0.89, P < 0.001) and revascularization (HR 0.86; 95% CI 0.80-0.93, P < 0.001), but no statistically significant difference across other endpoints. There were no significant differences in long-term outcomes for BA compared with BMS. CONCLUSIONS: There was lower mortality and reduced risk for MI, revascularization, and stroke, but a similar rate of bleeding with DES compared with other modalities. Our results indicate that DES use is a comparatively effective strategy to treat elderly patients with ISR.

Gastroesophageal signet ring cell carcinoma morbidity and mortality: A retrospective review.

BACKGROUND: Upper gastrointestinal (GI) signet ring cell carcinomas (SRCC) confer a poor prognosis. The benefit of operative intervention for this patient group is controversial in terms of overall survival. AIM: To investigate factors relating to survival in patients with upper GI SRCC. METHODS: A retrospective, tertiary, single-centre review of patients who were diagnosed with oesophageal, gastroesophageal junction and gastric SRCC was performed. The primary outcome was to compare mortality of patients who underwent operative management with those who had nonoperative management. Secondary outcomes included assessing the relationship between demographic and histopathological factors, and survival. RESULTS: One hundred and thirty-one patients were included. The one-year survival for the operative group was 81% and for the nonoperative group was 19.1%. The five-year survival in the operative group was 28.6% vs 1.5% in the nonoperative group. The difference in overall survival between groups was statistically significant (HR 0.19, 95%CI (0.13-0.30), P < 0.001). There was no difference in survival when adjusting for age, smoking status or gender. On multivariate analysis, patients who underwent surgical management, those with a lower stage of disease, and those with a lower Charlson Comorbidity Index (CCI) had significantly improved survival. CONCLUSION: Well-selected patients with upper GI SRCC appear to have reasonable medium-term survival following surgery. Offering surgery to a carefully selected patient group may improve the outcome for this disease.