Prognostic Importance of Axillary Lymph Node Response to Neoadjuvant Systemic Therapy on Axillary Surgery in Breast Cancer-A Single Center Experience.

Neoadjuvant systemic treatment (NST) is the standard treatment for HER2+, triple-negative (TN), and highly proliferative luminal HER2- early breast cancer. Pathologic complete response (pCR) after NST is associated with improved outcomes. We evaluated the predictive factors for axillary-pCR (AXpCR) and its impact on the extent of axillary node surgery. This retrospective study included 92 patients (median age of 50.4 years) with an initially node-positive disease. Patients were treated with molecular subtype-specific NST (4.3% were luminal A-like, 28.3% luminal HER2-, 26.1% luminal HER2+, 18.5% HER2+ non-luminal, and 22.8% TN). Axillary-, breast- and total-pCR were achieved in 52.2%, 48.9%, and 38% of patients, respectively. In a binary logistic regression model for the whole population, the only independent factor significantly associated with AXpCR was breast-pCR (OR 7.4; 95% CI 2.6-20.9; p < 0.001). In patients who achieved breast-pCR, aggressive subtypes (HER2+ and TN; OR 11.24) and clinical tumor stage (OR 0.10) had a significant impact on achieving AXpCR. Axillary lymph node dissection was avoided in 53.3% of patients. In conclusion, in node-positive patients with HER2+ and TN subtypes, who achieved breast-pCR after NST, de-escalation of axillary surgery could be considered in most cases.

Fast treatment of anterior shoulder dislocations with two sedation-free methods: The Davos self-reduction method and Arlt method.

Background: Various reduction techniques exist to treat traumatic shoulder dislocation, but best management remains unclear. Aims: To investigate the reduction rate of traumatic anteroinferior shoulder dislocations using two sedation-free techniques and success rates of subgroups. Methods: A single-center study was performed analysing shoulder dislocations in a two-year period. Adult patients with anteroinferior shoulder dislocation were included. Two sedation-free reduction techniques were used: the Davos self-reduction technique and the Arlt-chair technique. Two attempts were performed before sedation. All patients gave informed consent to study participation. Results: The investigated 106 patients (106 shoulder dislocations) had a mean age of 48 ± 18 years (74% male patients). The majority occurred during winter sports (76%). The overall success rate for both sedation-free reduction techniques was 82% (87 reduced shoulders, two attempts). A significantly increased success rate was found in patients without greater tuberosity fracture (86% without vs. 68% with fracture, p = 0.002) and for patients with repeated dislocation (93% vs. 80% for primary dislocation, p = 0.004). Time for reduction was 5 minutes (Davos technique) and 1 minute (Arlt-chair-technique). Associated injuries were mostly Hill Sachs lesions (78%). There was no major complication and no new-onset sensory deficit. Conclusion: Davos and Arlt reduction techniques allowed sedation-free and fast treatment for anteroinferior shoulder dislocation during winter sports.

Thoracoscopic Patch Repair of Congenital Diaphragmatic Hernia: Can Smaller Incisions Treat Larger Defects?

INTRODUCTION: Thoracoscopic CDH repair is increasingly performed for Type A and small Type B defects that are amenable to primary repair. However, the thoracoscopic approach is controversial for larger defects necessitating a patch due to technical complexity, intraoperative acidosis, and recurrence risk. We aim to compare clinical outcomes between thoracoscopic and open patch repair of Type B/C defects, using a standardized technique. METHODS: This is a single-center retrospective review of thoracoscopic and open CDH patch repairs January 2017-December 2021. We excluded primary repairs, Type D hernias, repairs on ECMO, recurrent repairs. Various preoperative, intraoperative, and postoperative variables were compared. Primary outcome was recurrence rate. Secondary outcomes included intraoperative pH and pCO2, operative time, and complication rates. RESULTS: Twenty-nine patients met inclusion criteria (open = 13, thoracoscopic = 16). The open cohort had lower o/e total fetal lung volume (29 vs 41.2%, p = 0.042), higher preoperative peak inspiratory pressures (24 vs 20 cm H2O, p = 0.007), were more frequently Type C defects (92.3 vs 31%, p = 0.002) and had liver "up" in left-sided hernias (46 vs 0%, p < 0.0001). Intraoperatively, median lowest pH and highest pCO2 did not differ; neither did overall median pH or pCO2. Operative times were similar (153 vs 194 min, p = 0.113). No difference in recurrence rates was identified, however postoperative complications were higher in the open group. There were no mortalities. CONCLUSIONS: Although we demonstrate higher disease severity of patients undergoing open repair, thoracoscopic patch repair for Type B/C defects is safe and effective in patients with favorable physiologic status, alleviating concerns for intraoperative acidosis, operative length, and risk of recurrence. LEVEL OF EVIDENCE: II.

Ovine Forestomach Matrix in the Surgical Management of Complex Lower-Extremity Soft-Tissue Defects.

BACKGROUND: Chronic lower-extremity defects may lead to major amputations and have severe consequences on patient quality of life and mortality. Dermal matrices have become part of the reconstructive ladder and are often deployed in these scenarios to quickly build neodermis, especially in volumetric defects over exposed bone and tendon initially, to allow for subsequent closure by means of split-thickness skin grafting (STSG) or secondary intention. Ovine forestomach matrix (OFM) is a decellularized extracellular matrix (ECM) bioscaffold available in both sheet and particulate forms that can be used as a dermal matrix in various soft-tissue reconstruction procedures. METHODS: This retrospective case series evaluated the use of OFM products in the surgical reconstruction of 50 cases (n = 50) comprised of challenging lower-extremity defects from seven healthcare centers. Patient records were reviewed to identify comorbidities, defect cause, defect size, presence of exposed structures, Centers for Disease Control and Prevention contamination score, Wagner grade, OFM graft use, time to 100% granulation tissue, STSG use, overall time to heal, and postoperative complications. The primary study outcomes were time (days) to 100% granulation tissue formation, with secondary outcomes including overall time to wound closure (weeks), STSG take at 1 week, and complications. RESULTS: The results of this case series demonstrate OFM as a clinically effective treatment in the surgical management of complex lower-extremity soft-tissue defects with exposed structures in patients with multiple comorbidities. One application of OFM products was effective in regenerating well-vascularized neodermis, often in the presence of exposed structures, with a mean time to 100% granulation of 26.0 ± 22.2 days. CONCLUSIONS: These data support the use of OFM as a safe, cost-effective, and clinically effective treatment option for coverage in complex soft-tissue wounds, including exposed vital structures, and to shorten the time to definitive wound closure in complicated patient populations.

The Structure of Maltooctaose-Bound Escherichia coli Branching Enzyme Suggests a Mechanism for Donor Chain Specificity.

Glycogen is the primary storage polysaccharide in bacteria and animals. It is a glucose polymer linked by α-1,4 glucose linkages and branched via α-1,6-linkages, with the latter reaction catalyzed by branching enzymes. Both the length and dispensation of these branches are critical in defining the structure, density, and relative bioavailability of the storage polysaccharide. Key to this is the specificity of branching enzymes because they define branch length. Herein, we report the crystal structure of the maltooctaose-bound branching enzyme from the enterobacteria E. coli. The structure identifies three new malto-oligosaccharide binding sites and confirms oligosaccharide binding in seven others, bringing the total number of oligosaccharide binding sites to twelve. In addition, the structure shows distinctly different binding in previously identified site I, with a substantially longer glucan chain ordered in the binding site. Using the donor oligosaccharide chain-bound Cyanothece branching enzyme structure as a guide, binding site I was identified as the likely binding surface for the extended donor chains that the E. coli branching enzyme is known to transfer. Furthermore, the structure suggests that analogous loops in branching enzymes from a diversity of organisms are responsible for branch chain length specificity. Together, these results suggest a possible mechanism for transfer chain specificity involving some of these surface binding sites.

Contralateral lymph node metastasis in recurrent ipsilateral breast cancer with Lynch syndrome: a locoregional event.

INTRODUCTION: Contralateral axillary lymph node metastasis (CALNM) in breast cancer (BC) is considered a distant metastasis, marking stage 4cancer. Therefore, it is generally treated as an incurable disease. However, in clinical practice, staging and treatment remain controversial due to a paucity of data, and the St. Gallen 2021 consensus panel recommended a curative approach in patients with oligometastatic disease. Aberrant lymph node (LN) drainage following previous surgery or radiotherapy is common. Therefore, CALNM may be considered a regional event rather than systemic disease, and a re-sentinel procedure aided by lymphoscintigraphy permits adequate regional staging. CASE REPORT: Here, we report a 37-year-old patient with Lynch syndrome who presented with CALNM in an ipsilateral relapse of a moderately differentiated invasive ductal BC (ER 90%, PR 30%, HER2 negative, Ki-67 25%, microsatellite stable), 3 years after the initial diagnosis. Lymphoscintigraphy detected a positive sentinel LN in the contralateral axilla despite no sign of LN involvement or distant metastases on FDG PET/CT or MRI. The patient underwent bilateral mastectomy with sentinel node dissection, surgical reconstruction with histological confirmation of the CALNM, left axillary dissection, adjuvant chemotherapy, and anti-hormone therapy. In addition to her regular BC follow-up visits, the patient will undergo annual colonoscopy, gastroscopy, abdominal, and vaginal ultrasound screening. In January 2023, the patient was free of progression for 23 months after initiation of treatment for recurrent BC and CALNM. CONCLUSION: This case highlights the value of delayed lymphoscintigraphy and the contribution of sentinel procedure for local control in the setting of recurrent BC. Aberrant lymph node drainage following previous surgery may be the underlying cause of CALNM. We propose that CALNM without evidence of systemic metastasis should be considered a regional event in recurrent BC, and thus, a curative approach can be pursued. The next AJCC BC staging should clarify the role of CALNM in recurrent BC to allow for the development of specific treatment guidelines.

Light controlled reversible Michael addition of cysteine: a new tool for dynamic site-specific labeling of proteins.

Cysteine-based Michael addition is a widely employed strategy for covalent conjugation of proteins, peptides, and drugs. The covalent reaction is irreversible in most cases, leading to a lack of control over the process. Utilizing spectroscopic analyses along with X-ray crystallographic studies, we demonstrate Michael addition of an engineered cysteine residue in human Cellular Retinol Binding Protein II (hCRBPII) with a coumarin analog that creates a non-fluorescent complex. UV-illumination reverses the conjugation, yielding a fluorescent species, presumably through a retro-Michael process. This series of events can be repeated between a bound and non-bound form of the cysteine reversibly, resulting in the ON-OFF control of fluorescence. The details of the mechanism of photoswitching was illuminated by recapitulation of the process in light irradiated single crystals, confirming the mechanism at atomic resolution.

Perioperative management with ferric carboxymaltose and tranexamic acid to reduce transfusion rate in gynaecological carcinoma surgery (TRANAFER-Study): study protocol for a single-blind, monocentre, randomised trial.

INTRODUCTION: Radical abdominal surgery is part of the standard treatment for women with advanced gynaecological carcinoma. The surgery often leads to intraoperative blood loss frequently exceeding 1000 mL. Approximately 50% of women undergoing radical surgery require blood transfusions. Perioperative blood transfusions have been shown to increase the risk of postoperative complications, delayed wound healing, increased length of stay, increased postoperative morbidity and mortality. Previous studies have demonstrated an association between perioperative anaemia and surgical morbidity and mortality. By reducing transfusions and improving recovery from surgery, preoperative diagnostic and management of perioperative anaemia is a great opportunity to optimise postoperative patient outcome. METHODS AND ANALYSIS: This is a single-blind, monocentre, randomised trial with four parallel groups (three therapeutic groups and one control group without treatment according to current standards of care) conducted in women undergoing radical gynaecological surgery. The primary study objective is to determine the effect of perioperative treatment with either intravenous iron, tranexamic acid or with a combination of both medicines on the reduction of intraoperative and postoperative red blood cell transfusions in gynaecological carcinoma patients. A total of N=126 women with gynaecological carcinoma will be recruited at the University Hospital Basel, Department of Gynaecology. Blood parameters will be measured at the recruitment, prior to surgery, 2 days after surgery and on the 21st-28th day after surgery. Recruitment started in August 2021. ETHICS AND DISSEMINATION: The study will be performed according to the guidelines of the Declaration of Helsinki and is approved by the Ethics Committee for Northwest and Central Switzerland in Basel (EKNZ Protocol ID 2020-01194). The results of this study will be published and presented in various scientific forums. TRIAL REGISTRATION NUMBER: NCT03792464.

A structural explanation for the mechanism and specificity of plant branching enzymes I and IIb.

Branching enzymes (BEs) are essential in the biosynthesis of starch and glycogen and play critical roles in determining the fine structure of these polymers. The substrates of these BEs are long carbohydrate chains that interact with these enzymes via multiple binding sites on the enzyme's surface. By controlling the branched-chain length distribution, BEs can mediate the physiological properties of starch and glycogen moieties; however, the mechanism and structural determinants of this specificity remain mysterious. In this study, we identify a large dodecaose binding surface on rice BE I (BEI) that reaches from the outside of the active site to the active site of the enzyme. Mutagenesis activity assays confirm the importance of this binding site in enzyme catalysis, from which we conclude that it is likely the acceptor chain binding site. Comparison of the structures of BE from Cyanothece and BE1 from rice allowed us to model the location of the donor-binding site. We also identified two loops that likely interact with the donor chain and whose sequences diverge between plant BE1, which tends to transfer longer chains, and BEIIb, which transfers exclusively much shorter chains. When the sequences of these loops were swapped with the BEIIb sequence, rice BE1 also became a short-chain transferring enzyme, demonstrating the key role these loops play in specificity. Taken together, these results provide a more complete picture of the structure, selectivity, and activity of BEs.

Comparison of 2D 4K vs. 3D HD laparoscopic imaging systems using a pelvitrainer model: a randomized controlled study.

Laparoscopic surgery provides well-known benefits, but it has technological limitations. Depth perception is particularly crucial, with three-dimensional (3D) imaging being superior to two-dimensional (2D) HD imaging. However, with the introduction of 4K resolution monitors, 2D rendering is capable of providing higher-quality visuals. Therefore, this study aimed to compare 3D HD and 2D 4K imaging using a pelvitrainer model. Eight experts and 32 medical students were performing the same four standardized tasks using 2D 4K and 3D HD imaging systems. Task completion time and the number of errors made were recorded. The Wilcoxon test and mixed-effects models were used to analyze the results. Students were significantly faster in all four tasks when using the 3D HD perspective. The median difference ranged from 18 s in task 3 (P < 0.003) up to 177.5 s in task 4 (P < 0.001). With the exception of task 4, students demonstrated significantly fewer errors in all tasks involving 3D HD imaging. The experts' results confirmed these findings, as they were also faster in all four tasks using 3D HD, which was significant for task 1 (P < 0.001) and task 4 (P < 0.006). The expert group also achieved better movement accuracy using the 3D HD system, with fewer mistakes made in all four tasks, which was significant in task 4 (P < 0.001). Participants in both groups achieved better results with the 3D HD imaging system than with the 2D 4K system. The 3D HD image system should be used when available. Trial registration: this trial is registered at research registry under the identifier researchregistry6852.

Cg10062 Catalysis Forges a Link between Acetylenecarboxylic Acid and Bacterial Metabolism.

The reliance of biocatalysis on plant-derived carbon for the synthesis of fuels and chemicals places it in direct competition with food production for resources. A potential solution to this problem is development of a metabolic link between alternative carbon sources and bacterial metabolism. Acetylenecarboxylic acid, which can be synthesized from methane and carbon dioxide, could enable this connection. It was previously shown that the enzyme Cg10062 catalyzes hydration of acetylenecarboxylate to afford malonate semialdehyde. Subsequent hydration-dependent decarboxylation to form acetaldehyde (81%), which was also observed, limits its biocatalytic usefulness. Several Cg10062 variants including E114Q and E114D do not catalyze decarboxylation and provide malonate semialdehyde as the sole product, albeit with substantially reduced catalytic activity. To identify an efficient enzyme capable of catalyzing acetylenecarboxylate hydration without decarboxylation, we undertook a mechanistic investigation of Cg10062 using mutagenesis, kinetic characterization, and X-ray crystallography. Cg10062 is a member of the tautomerase superfamily of enzymes, characterized by their ß-α-ß protein fold and an N-terminal proline residue situated at the center of the enzyme active site. Along with Pro-1, five additional active site residues (His-28, Arg-70, Arg-73, Tyr-103, and Glu-114) are required for Cg10062 activity. Incubation of crystals of four catalytically slow variants of Cg10062 with acetylenecarboxylate resulted in atomic resolution structures of Pro-1 bound to a complete set of intermediates, fully elaborating the detailed mechanism of the enzyme and establishing the process to involve covalent catalysis. Further, the intermediate-bound E114D structure explains the mechanism governing decarboxylation suppression. Together, these studies provide the most detailed picture of the catalytic mechanism of a tautomerase enzyme to date.

Control of Protonated Schiff Base Excited State Decay within Visual Protein Mimics: A Unified Model for Retinal Chromophores.

Artificial biomimetic chromophore-protein complexes inspired by natural visual pigments can feature color tunability across the full visible spectrum. However, control of excited state dynamics of the retinal chromophore, which is of paramount importance for technological applications, is lacking due to its complex and subtle photophysics/photochemistry. Here, ultrafast transient absorption spectroscopy and quantum mechanics/molecular mechanics simulations are combined for the study of highly tunable rhodopsin mimics, as compared to retinal chromophores in solution. Conical intersections and transient fluorescent intermediates are identified with atomistic resolution, providing unambiguous assignment of their ultrafast excited state absorption features. The results point out that the electrostatic environment of the chromophore, modified by protein point mutations, affects its excited state properties allowing control of its photophysics with same power of chemical modifications of the chromophore. The complex nature of such fine control is a fundamental knowledge for the design of bio-mimetic opto-electronic and photonic devices.

Design of Large Stokes Shift Fluorescent Proteins Based on Excited State Proton Transfer of an Engineered Photobase.

The incredible potential for fluorescent proteins to revolutionize biology has inspired the development of a variety of design strategies to address an equally broad range of photophysical characteristics, depending on potential applications. Of these, fluorescent proteins that simultaneously exhibit high quantum yield, red-shifted emission, and wide separation between excitation and emission wavelengths (Large Stokes Shift, LSS) are rare. The pursuit of LSS systems has led to the formation of a complex, obtained from the marriage of a rationally engineered protein (human cellular retinol binding protein II, hCRBPII) and different fluorogenic molecules, capable of supporting photobase activity. The large increase in basicity upon photoexcitation leads to protonation of the fluorophore in the excited state, dramatically red-shifting its emission, leading to an LSS protein/fluorophore complex. Essential for selective photobase activity is the intimate involvement of the target protein structure and sequence that enables Excited State Proton Transfer (ESPT). The potential power and usefulness of the strategy was demonstrated in live cell imaging of human cell lines.

Single-stage restorative proctocolectomy for ulcerative colitis in pediatric patients: a safe alternative.

BACKGROUND: Surgical management for refractory ulcerative colitis (UC) has been restorative proctocolectomy (RP) with ileal-pouch-anal-anastomosis (IPAA) done as one to three stages, with safety and effectiveness of a single-stage operation unclear. METHODS: Pediatric UC patients from 2004 to 2019 who underwent RP/IPAA in the initial operation were retrospectively reviewed. 1-stage operations were matched 1:2 to 2-stage operations using age, duration of disease, and disease severity. RESULTS: Ninety-nine patients (33 1-stage, 66 2-stage) were identified. The median total operative time was shorter in the 1-stage group (6 h:00 min vs. 7 h:47 min, p = 0.004). Total length of stay was shorter in the 1-stage group (9 vs. 17 days, p = 0.001). Rates of readmission were higher in 2-stage group (30 vs. 9%, p = 0.02). There was no difference in pouch leak rates (p = 1.00). Stricture rates were higher in the 2-stage group (50 vs. 16%, p = 0.005). Functional outcomes including pouchitis (p = 0.13), daily bowel movements (p = 0.37), and incontinence (p = 0.77) were all similar. CONCLUSIONS: Restorative proctocolectomy with IPAA in children with UC can be performed as a 1- or 2-stage operation with equivalent short-term, long-term, and functional outcomes in similar risk population. Our findings suggest 1-stage RP/IPAA operations without ileostomy are a safe alternative for patients considered for a 2-stage operation.

A hydrophobic residue stabilizes dimers of regulatory ACT-like domains in plant basic helix-loop-helix transcription factors.

About a third of the plant basic helix-loop-helix (bHLH) transcription factors harbor a C-terminal aspartate kinase, chorismate mutase, and TyrA (ACT)-like domain, which was originally identified in the maize R regulator of anthocyanin biosynthesis, where it modulates the ability of the bHLH to dimerize and bind DNA. Characterization of other bHLH ACT-like domains, such as the one in the Arabidopsis R ortholog, GL3, has not definitively confirmed dimerization, raising the question of the overall role of this potential regulatory domain. To learn more, we compared the dimerization of the ACT-like domains of R (RACT) and GL3 (GL3ACT). We show that RACT dimerizes with a dissociation constant around 100 nM, over an order of magnitude stronger than GL3ACT. Structural predictions combined with mutational analyses demonstrated that V568, located in a hydrophobic pocket in RACT, is important: when mutated to the Ser residue present in GL3ACT, dimerization affinity dropped by almost an order of magnitude. The converse S595V mutation in GL3ACT significantly increased the dimerization strength. We cloned and assayed dimerization for all identified maize ACT-like domains and determined that 12 of 42 formed heterodimers in yeast two-hybrid assays, irrespective of whether they harbored V568, which was often replaced by other aliphatic amino acids. Moreover, we determined that the presence of polar residues at that position occurs only in a small subset of anthocyanin regulators. The combined results provide new insights into possibly regulatory mechanisms and suggest that many of the other plant ACT-like domains associate to modulate fundamental cellular processes.

Does the timing of pouch creation in 2-stage operations for pediatric patients with ulcerative colitis matter?

INTRODUCTION: Children with fulminant ulcerative colitis(UC) traditionally undergo 2-stage operations: restorative-proctocolectomy(RP/IPAA) and ileostomy followed by ostomy closure. In the biologic era, surgeons have modified their strategy: initial subtotal-colectomy/diversion, followed by RP/IPAA without diversion. Yet, evidence on efficacy and functional outcomes with the "modified 2-stage" approach is limited in children. We sought to compare the timing of pouch creation in 2-stage operations to determine outcomes. METHODS: This is a retrospective study of children with UC undergoing either a traditional 2-stage RP/IPAA or modified 2-stage RP/IPAA between 2010 and 2019. Complications (leak, stricture, wound-infection) were recorded at 90-days and 1 year from 2nd operation. RESULTS: N = 57 (Traditional n = 40, Modified n = 17). Median time to surgery from consultation was shorter in the modified-group (7 vs.25 days, p = 0.01). Preoperatively, the modified-group had lower albumin(p = 0.01), higher CRP(p = 0.01), and more frequently took biologics within 90-daysp=0.001). After re-establishing intestinal continuity, stricture requiring dilation was higher in the traditional-group (59% vs.18%, p = 0.008). No difference in pouch leak (p = 0.38), bowel obstruction(p = 0.35), loperamide dose(p = 0.21), or incontinence(p = 0.38) was observed. CONCLUSION: Delaying pouch creation to the second operation without a protective ileostomy as a modified 2-stage is safe in a sicker and more acute pediatric population.

Evidence that specific interactions play a role in the cholesterol sensitivity of G protein-coupled receptors.

G protein-coupled receptors (GPCRs) are known to be modulated by membrane cholesterol levels, but whether or not the effects are caused by specific receptor-cholesterol interactions or cholesterol's general effects on the membrane is not well-understood. We performed coarse-grained molecular dynamics (CGMD) simulations coupled with structural bioinformatics approaches on the ß2-adrenergic receptor (ß2AR) and the cholecystokinin (CCK) receptor subfamily. The ß2AR has been shown to be sensitive to membrane cholesterol and cholesterol molecules have been clearly resolved in numerous ß2AR crystal structures. The two CCK receptors are highly homologous and preserve similar cholesterol recognition motifs but despite their homology, CCK1R shows functional sensitivity to membrane cholesterol while CCK2R does not. Our results offer new insights into how cholesterol modulates GPCR function by showing cholesterol interactions with ß2AR that agree with previously published data; additionally, we observe differential and specific cholesterol binding in the CCK receptor subfamily while revealing a previously unreported Cholesterol Recognition Amino-acid Consensus (CRAC) sequence that is also conserved across 38% of class A GPCRs. A thermal denaturation assay (LCP-Tm) shows that mutation of a conserved CRAC sequence on TM7 of the ß2AR affects cholesterol stabilization of the receptor in a lipid bilayer. The results of this study provide a better understanding of receptor-cholesterol interactions that can contribute to novel and improved therapeutics for a variety of diseases.

Harnessing the power of an X-ray laser for serial crystallography of membrane proteins crystallized in lipidic cubic phase.

Serial femtosecond crystallography (SFX) with X-ray free-electron lasers (XFELs) has proven highly successful for structure determination of challenging membrane proteins crystallized in lipidic cubic phase; however, like most techniques, it has limitations. Here we attempt to address some of these limitations related to the use of a vacuum chamber and the need for attenuation of the XFEL beam, in order to further improve the efficiency of this method. Using an optimized SFX experimental setup in a helium atmosphere, the room-temperature structure of the adenosine A2A receptor (A2AAR) at 2.0 Šresolution is determined and compared with previous A2AAR structures determined in vacuum and/or at cryogenic temperatures. Specifically, the capability of utilizing high XFEL beam transmissions is demonstrated, in conjunction with a high dynamic range detector, to collect high-resolution SFX data while reducing crystalline material consumption and shortening the collection time required for a complete dataset. The experimental setup presented herein can be applied to future SFX applications for protein nanocrystal samples to aid in structure-based discovery efforts of therapeutic targets that are difficult to crystallize.