Selective Serotonin Reuptake Inhibitors within Cells: Temporal Resolution in Cytoplasm, Endoplasmic Reticulum, and Membrane.

Selective serotonin reuptake inhibitors (SSRIs) are the most prescribed treatment for individuals experiencing major depressive disorder. The therapeutic mechanisms that take place before, during, or after SSRIs bind the serotonin transporter (SERT) are poorly understood, partially because no studies exist on the cellular and subcellular pharmacokinetic properties of SSRIs in living cells. We studied escitalopram and fluoxetine using new intensity-based, drug-sensing fluorescent reporters targeted to the plasma membrane, cytoplasm, or endoplasmic reticulum (ER) of cultured neurons and mammalian cell lines. We also used chemical detection of drug within cells and phospholipid membranes. The drugs attain equilibrium in neuronal cytoplasm and ER at approximately the same concentration as the externally applied solution, with time constants of a few s (escitalopram) or 200-300 s (fluoxetine). Simultaneously, the drugs accumulate within lipid membranes by ≥18-fold (escitalopram) or 180-fold (fluoxetine), and possibly by much larger factors. Both drugs leave cytoplasm, lumen, and membranes just as quickly during washout. We synthesized membrane-impermeant quaternary amine derivatives of the two SSRIs. The quaternary derivatives are substantially excluded from membrane, cytoplasm, and ER for >2.4 h. They inhibit SERT transport-associated currents sixfold or 11-fold less potently than the SSRIs (escitalopram or fluoxetine derivative, respectively), providing useful probes for distinguishing compartmentalized SSRI effects. Although our measurements are orders of magnitude faster than the therapeutic lag of SSRIs, these data suggest that SSRI-SERT interactions within organelles or membranes may play roles during either the therapeutic effects or the antidepressant discontinuation syndrome.SIGNIFICANCE STATEMENT Selective serotonin reuptake inhibitors stabilize mood in several disorders. In general, these drugs bind to SERT, which clears serotonin from CNS and peripheral tissues. SERT ligands are effective and relatively safe; primary care practitioners often prescribe them. However, they have several side effects and require 2-6 weeks of continuous administration until they act effectively. How they work remains perplexing, contrasting with earlier assumptions that the therapeutic mechanism involves SERT inhibition followed by increased extracellular serotonin levels. This study establishes that two SERT ligands, fluoxetine and escitalopram, enter neurons within minutes, while simultaneously accumulating in many membranes. Such knowledge will motivate future research, hopefully revealing where and how SERT ligands engage their therapeutic target(s).

Metabolic Outcomes After Pancreas Transplant Alone From Donation After Circulatory Death Donors-The UK Transplant Registry Analysis.

Extrapolating data from early DCD (donation after circulatory death) kidney transplantation, pancreas transplants from DCD grafts were feared to have worse metabolic outcomes. Hence, we aimed to address the question of pancreas transplant alone (PTA) from DCD donors-are our concerns justified? A UK transplant registry analysis of 185 PTA performed between 2005 and 2018 was done. All early graft losses (<3 months) were excluded to allow focus on the metabolic outcomes (HbA1c, weight gain and incidence of secondary diabetic macrovascular complications). The aim was to compare the metabolic outcomes, rejection rates (including the need for steroids), patient and graft survival between DBD (Donation after brainstem death) and DCD groups. After excluding early graft losses, data from 162 PTA (DBD = 114 and DCD = 48) were analyzed. Body mass index of the donor was less in DCD group (DBD = 23.40 vs. DCD = 22.25, p = 0.006) and the rest of the baseline transplant characteristics were comparable. There were no significant differences in the HbA1c, weight gain, rejection rate, and incidence of secondary diabetic macrovascular complications post-transplant between DBD and DCD recipients. The 1-, 5-, and 10-year patient and graft survival were similar in both the groups. PTA from DCD donors have equivalent metabolic outcomes and survival (patient/graft) as that of DBD donors.

Three Mutations Convert the Selectivity of a Protein Sensor from Nicotinic Agonists to S-Methadone for Use in Cells, Organelles, and Biofluids.

We report a reagentless, intensity-based S-methadone fluorescent sensor, iS-methadoneSnFR, consisting of a circularly permuted GFP inserted within the sequence of a mutated bacterial periplasmic binding protein (PBP). We evolved a previously reported nicotine-binding PBP to become a selective S-methadone-binding sensor, via three mutations in the PBP's second shell and hinge regions. iS-methadoneSnFR displays the necessary sensitivity, kinetics, and selectivity─notably enantioselectivity against R-methadone─for biological applications. Robust iS-methadoneSnFR responses in human sweat and saliva and mouse serum enable diagnostic uses. Expression and imaging in mammalian cells demonstrate that S-methadone enters at least two organelles and undergoes acid trapping in the Golgi apparatus, where opioid receptors can signal. This work shows a straightforward strategy in adapting existing PBPs to serve real-time applications ranging from subcellular to personal pharmacokinetics.

Pancreas Transplantation in Black, Asian and Minority Ethnic Patients-Single Centre Experience in the UK.

Ethnic disparities in the outcomes after simultaneous pancreas kidney (SPK) transplantation still exist. The influence of ethnicity on the outcomes of pancreas transplantation in the UK has not been reported and hence we aimed to investigate our cohort. A retrospective analysis of all pancreas transplant recipients (n = 171; Caucasians = 118/Black Asian Ethnic Minorities, BAME = 53) from 2006 to 2020 was done. The median follow-up was 80 months. Patient & pancreas graft survival, rejection rate, steroid free maintenance rate, HbA1c, weight gain, and the incidence of secondary diabetic complications post-transplant were compared between the groups. p < 0.003 was considered significant (corrected for multiple hypothesis testing). Immunosuppression consisted of alemtuzumab induction and steroid free maintenance with tacrolimus and mycophenolate mofetil. Pancreas graft & patient survival were equivalent in both the groups. BAME recipients had a higher prevalence of type-2 diabetes mellitus pre-transplant (BAME = 30.19% vs. Caucasians = 0.85%, p < 0.0001), and waited for a similar time to transplantation once waitlisted, although pre-emptive SPK transplantation rate was higher for Caucasian recipients (Caucasians = 78.5% vs. BAME = 0.85%, p < 0.0001). Despite equivalent rejections & steroid usage, BAME recipients gained more weight (BAME = 7.7% vs. Caucasians = 1.8%, p = 0.001), but had similar HbA1c (functioning grafts) at 3-,12-, 36-, and 60-months post-transplant.

HoloMentor: A Novel Mixed Reality Surgical Anatomy Curriculum for Robot-Assisted Radical Prostatectomy.

OBJECTIVES: The disruption to surgical training and medical education caused by the global COVID-19 pandemic highlighted the need for realistic, reliable, and engaging educational opportunities available outside of the operating theatre and accessible for trainees of all levels. This article presents the design and development of a virtual reality curriculum which simulates the surgical mentorship experience outside of the operating theatre, with a focus on surgical anatomy and surgical decision-making. METHOD: This was a multi-institutional study between London's King's College and Imperial College. The index procedure selected for the module was robotic radical prostatectomy. For each stage of the surgical procedure, subject-matter experts (N = 3) at King's College London, identified (1) the critical surgical-decision making points, (2) critical anatomical landmarks, and (3) tips and techniques for overcoming intraoperative challenges. Content validity was determined by an independent panel of subject-matter experts (N = 8) at Imperial College, London, using Fleiss' kappa statistic. The experts' teaching points were combined with operative footage and illustrative animations, and projected onto a virtual reality headset. The module was piloted to surgical science students (N = 15). Quantitative analysis compared participants' confidence regarding their anatomical knowledge before and after taking the module. Qualitative data were gathered from students regarding their views on using the virtual reality model. RESULTS: Multi-rater agreement between experts was above the 70.0% threshold for all steps of the procedure. Seventy-three percentage of pilot study participants "agreed" or "strongly agreed" that they achieved a better understanding of surgical anatomy and the rationale behind each procedural step. This was reflected in an increase in the median knowledge score after trialing the curriculum (p < 0.001). 100% of subject-matter experts and 93.3% of participants "agreed" or "strongly agreed" that virtual mentorship would be useful for future surgical training. CONCLUSIONS: This study demonstrated that virtual surgical mentorship could be a feasible and cost-effective alternative to traditional training methods with the potential to improve technical skills, such as operative proficiency and nontechnical skills such as decision-making and situational judgement.

A general method to fine-tune fluorophores for live-cell and in vivo imaging.

Pushing the frontier of fluorescence microscopy requires the design of enhanced fluorophores with finely tuned properties. We recently discovered that incorporation of four-membered azetidine rings into classic fluorophore structures elicits substantial increases in brightness and photostability, resulting in the Janelia Fluor (JF) series of dyes. We refined and extended this strategy, finding that incorporation of 3-substituted azetidine groups allows rational tuning of the spectral and chemical properties of rhodamine dyes with unprecedented precision. This strategy allowed us to establish principles for fine-tuning the properties of fluorophores and to develop a palette of new fluorescent and fluorogenic labels with excitation ranging from blue to the far-red. Our results demonstrate the versatility of these new dyes in cells, tissues and animals.

Bright photoactivatable fluorophores for single-molecule imaging.

Small-molecule fluorophores are important tools for advanced imaging experiments. We previously reported a general method to improve small, cell-permeable fluorophores which resulted in the azetidine-containing 'Janelia Fluor' (JF) dyes. Here, we refine and extend the utility of these dyes by synthesizing photoactivatable derivatives that are compatible with live-cell labeling strategies. Once activated, these derived compounds retain the superior brightness and photostability of the JF dyes, enabling improved single-particle tracking and facile localization microscopy experiments.

Specific modulation of protein activity by using a bioorthogonal reaction.

Unnatural amino acids with bioorthogonal reactive groups have the potential to provide a rapid and specific mechanism for covalently inhibiting a protein of interest. Here, we use mutagenesis to insert an unnatural amino acid containing an azide group (Z) into the target protein at positions such that a "click" reaction with an alkyne modulator (X) will alter the function of the protein. This bioorthogonally reactive pair can engender specificity of X for the Z-containing protein, even if the target is otherwise identical to another protein, allowing for rapid target validation in living cells. We demonstrate our method using inhibition of the Escherichia coli enzyme aminoacyl transferase by both active-site occlusion and allosteric mechanisms. We have termed this a "clickable magic bullet" strategy, and it should be generally applicable to studying the effects of protein inhibition, within the limits of unnatural amino acid mutagenesis.

Efficient synthesis and in vivo incorporation of acridon-2-ylalanine, a fluorescent amino acid for lifetime and Förster resonance energy transfer/luminescence resonance energy transfer studies.

The amino acid acridon-2-ylalanine (Acd) can be a valuable probe of protein conformational change because it is a long lifetime, visible wavelength fluorophore that is small enough to be incorporated during ribosomal biosynthesis. Incorporation of Acd into proteins expressed in Escherichia coli requires efficient chemical synthesis to produce large quantities of the amino acid and the generation of a mutant aminoacyl tRNA synthetase that can selectively charge the amino acid onto a tRNA. Here, we report the synthesis of Acd in 87% yield over five steps from Tyr and the identification of an Acd synthetase by screening candidate enzymes previously evolved from Methanococcus janaschii Tyr synthetase for unnatural amino acid incorporation. Furthermore, we characterize the photophysical properties of Acd, including quenching interactions with select natural amino acids and Förster resonance energy transfer (FRET) interactions with common fluorophores such as methoxycoumarin (Mcm). Finally, we demonstrate the value of incorporation of Acd into proteins, using changes in Acd fluorescence lifetimes, Mcm/Acd FRET, or energy transfer to Eu(3+) to monitor protein folding and binding interactions.

Fluorescence Screens for Identifying Central Nervous System-Acting Drug-Biosensor Pairs for Subcellular and Supracellular Pharmacokinetics.

Subcellular pharmacokinetic measurements have informed the study of central nervous system (CNS)-acting drug mechanisms. Recent investigations have been enhanced by the use of genetically encoded fluorescent biosensors for drugs of interest at the plasma membrane and in organelles. We describe screening and validation protocols for identifying hit pairs comprising a drug and biosensor, with each screen including 13-18 candidate biosensors and 44-84 candidate drugs. After a favorable hit pair is identified and validated via these protocols, the biosensor is then optimized, as described in other papers, for sensitivity and selectivity to the drug. We also show sample hit pair data that may lead to future intensity-based drug-sensing fluorescent reporters (iDrugSnFRs). These protocols will assist scientists to use fluorescence responses as criteria in identifying favorable fluorescent biosensor variants for CNS-acting drugs that presently have no corresponding biosensor partner. This protocol was validated in: eLife (2022), DOI: 10.7554/eLife.74648 Graphical abstract.

Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands.

Nicotinic partial agonists provide an accepted aid for smoking cessation and thus contribute to decreasing tobacco-related disease. Improved drugs constitute a continued area of study. However, there remains no reductionist method to examine the cellular and subcellular pharmacokinetic properties of these compounds in living cells. Here, we developed new intensity-based drug-sensing fluorescent reporters (iDrugSnFRs) for the nicotinic partial agonists dianicline, cytisine, and two cytisine derivatives - 10-fluorocytisine and 9-bromo-10-ethylcytisine. We report the first atomic-scale structures of liganded periplasmic binding protein-based biosensors, accelerating development of iDrugSnFRs and also explaining the activation mechanism. The nicotinic iDrugSnFRs detect their drug partners in solution, as well as at the plasma membrane (PM) and in the endoplasmic reticulum (ER) of cell lines and mouse hippocampal neurons. At the PM, the speed of solution changes limits the growth and decay rates of the fluorescence response in almost all cases. In contrast, we found that rates of membrane crossing differ among these nicotinic drugs by >30-fold. The new nicotinic iDrugSnFRs provide insight into the real-time pharmacokinetic properties of nicotinic agonists and provide a methodology whereby iDrugSnFRs can inform both pharmaceutical neuroscience and addiction neuroscience.

Expanding the donor pool in pancreas transplantation.

PURPOSE OF REVIEW: The current era of organ shortage has necessitated expansion of the currently available organ donor pool, to increase the number of pancreases available for transplant. This review summarizes the cumulative efforts of various centers in making this possible. RECENT FINDINGS: Various centers are consistently reporting their experience with marginal donors; recent additions to the cohort have been increase in pancreases from donors after cardiac death (controlled and uncontrolled), update on long-term outcomes of live pancreas donors, as well as efforts at objectively assessing donor risk. SUMMARY: It has become important for the transplanting surgeon to make difficult decisions on organ suitability and appropriateness depending upon the recipient's status. Further more, limiting further damage to these vulnerable grafts is important in improving utilization as well as successful transplantation.

Imaging in whole organ pancreatic transplants and a multimodality review of its complications.

This pictorial review will describe the normal anatomy of whole organ pancreatic transplants and the common surgical variants with which the radiologist should be familiar. Complications may be divided into (1) vascular: arterial occlusion and stenosis, venous thrombosis, pseudoaneurysms and arteriovenous fistulae, (2) parenchymal complications such as pancreatitis and the variety of peripancreatic collections, and (3) enteric complications including leak and fistula formation. The radiologist plays a crucial role in the initial assessment of graft anatomy and perfusion, prompt diagnosis, and increasingly, in the management of complications.

Gastrointestinal Bleeding in a Pancreas Transplant Recipient: A Case to Remember.

BACKGROUND Pancreas transplantation has proven to be the most effective therapeutic option for insulin-dependent diabetes mellitus. However, despite advances in surgical technique and continuously improving outcomes, pancreas transplantation has the highest complication rate among all solid-organ transplants. Vascular complications in particular can be catastrophic, with graft- and life-threatening potential. Ectopic variceal bleeding is less common and is rarely reported in the literature. CASE REPORT A 51-year-old man presented with recurrent intermittent gastrointestinal bleeding (GIB) associated with hepatic dysfunction and portal hypertension 4 years after a successful pancreas-after-kidney transplant. Apart from positive serology for hepatitis E virus, all the other liver disease screening results were negative. He was extensively investigated with 6 computed tomography (CT) scans, 3 esophago-gastro-duodenoscopies (EGD), 3 colonoscopies, and 1 visceral arteriogram before the plausible diagnosis of ectopic trans-anastomotic variceal bleeding involving the pancreas transplant was established. Selective variceal catheterization and embolization were done with 3% sodium tetradecyl sulphate (STD). He remained free of bleeding after embolization. CONCLUSIONS This case report adds to the scanty literature on the management of ectopic variceal bleeding in a pancreas transplant recipient. Diagnosis of ectopic varix is usually challenging and frequently requires a visceral arteriogram. We describe a novel minimally-interventional technique to obtain source control and also discuss the complexity involved in the management, along with future implications.

Transfusion-Dependent Anemia in a Simultaneous Pancreas and Kidney Transplant Recipient.

A case of transfusion-dependent anemia in a simultaneous pancreas and kidney (SPK) transplant recipient that masqueraded as gastrointestinal bleeding (GIB) is described. The anemia was attributed to bleeding from the donor duodenal cuff based on balloon enteroscopy findings. The patient underwent multiple contrast-enhanced computed tomography scans and multiple endoscopies with confounding features until, eventually, the diagnosis was established. We discuss the diagnostic difficulties and the therapeutic dilemma, along with the pitfalls in ascertaining the final diagnosis.

Cohort Study of Outpatient Hemodialysis Management Strategies for COVID-19 in North-West London.

BACKGROUND: Dialysis patients are at risk of severe coronavirus disease 2019 (COVID-19). We managed COVID-19 hemodialysis outpatients in dedicated satellite dialysis units. This provided rare opportunity to study early disease progress in community-based patients. We aimed to (i) understand COVID-19 progression, (ii) identify markers of future clinical severity, and (iii) assess associations between dialysis management strategies and COVID-19 clinical outcomes. METHODS: We conducted a cohort study of all outpatients managed at a COVID-19 hemodialysis unit. We analyzed data recorded as part of providing COVID-19 clinical care. We analyzed associations between features at diagnosis and the first 3 consecutive hemodialysis sessions in patients who required future hospital admission, and those who had died at 28 days. RESULTS: Isolated outpatient hemodialysis was provided to 106 patients over 8 weeks. No patients received antiviral medication or hydroxychloroquine. Twenty-one patients (20%) were admitted at COVID-19 diagnosis; 29 of 85 patients (34%) were admitted after initial outpatient management; 16 patients (15%) died. By multivariate analysis, nonactive transplant list status, use of institutional transport, and increased white cell count associated with future hospitalization and increased age associated with death. Oxygen saturations progressively decreased over the first 3 dialysis sessions in the cohorts that progressed to future hospital admission or death. Mean ultrafiltration volume of the first 3 hemodialysis sessions was reduced in the same cohorts. CONCLUSIONS: Outpatient hemodialysis in patients with COVID-19 is safe for patients and staff. Features at the first 3 dialysis sessions can identify individuals at risk of future hospitalization and death from COVID-19.

Anticoagulation in simultaneous pancreas kidney transplantation - On what basis?

BACKGROUND: Despite technical refinements, early pancreas graft loss due to thrombosis continues to occur. Conventional coagulation tests (CCT) do not detect hypercoagulability and hence the hypercoagulable state due to diabetes is left untreated. Thromboelastogram (TEG) is an in-vitro diagnostic test which is used in liver transplantation, and in various intensive care settings to guide anticoagulation. TEG is better than CCT because it is dynamic and provides a global hemostatic profile including fibrinolysis. AIM: To compare the outcomes between TEG and CCT (prothrombin time, activated partial thromboplastin time and international normalized ratio) directed anticoagulation in simultaneous pancreas and kidney (SPK) transplant recipients. METHODS: A single center retrospective analysis comparing the outcomes between TEG and CCT-directed anticoagulation in SPK recipients, who were matched for donor age and graft type (donors after brainstem death and donors after circulatory death). Anticoagulation consisted of intravenous (IV) heparin titrated up to a maximum of 500 IU/h based on CCT in conjunction with various clinical parameters or directed by TEG results. Graft loss due to thrombosis, anticoagulation related bleeding, radiological incidence of partial thrombi in the pancreas graft, thrombus resolution rate after anticoagulation dose escalation, length of the hospital stays and, 1-year pancreas and kidney graft survival between the two groups were compared. RESULTS: Seventeen patients who received TEG-directed anticoagulation were compared against 51 contemporaneous SPK recipients (ratio of 1: 3) who were anticoagulated based on CCT. No graft losses occurred in the TEG group, whereas 11 grafts (7 pancreases and 4 kidneys) were lost due to thrombosis in the CCT group (P = 0.06, Fisher's exact test). The overall incidence of anticoagulation related bleeding (hematoma/ gastrointestinal bleeding/ hematuria/ nose bleeding/ re-exploration for bleeding/ post-operative blood transfusion) was 17.65% in the TEG group and 45.10% in the CCT group (P = 0.05, Fisher's exact test). The incidence of radiologically confirmed partial thrombus in pancreas allograft was 41.18% in the TEG and 25.50% in the CCT group (P = 0.23, Fisher's exact test). All recipients with partial thrombi detected in computed tomography (CT) scan had an anticoagulation dose escalation. The thrombus resolution rates in subsequent scan were 85.71% and 63.64% in the TEG group vs the CCT group (P = 0.59, Fisher's exact test). The TEG group had reduced blood product usage {10 packed red blood cell (PRBC) and 2 fresh frozen plasma (FFP)} compared to the CCT group (71 PRBC/ 10 FFP/ 2 cryoprecipitate and 2 platelets). The proportion of patients requiring transfusion in the TEG group was 17.65% vs 39.25% in the CCT group (P = 0.14, Fisher's exact test). The median length of hospital stay was 18 days in the TEG group vs 31 days in the CCT group (P = 0.03, Mann Whitney test). The 1-year pancreas graft survival was 100% in the TEG group vs 82.35% in the CCT group (P = 0.07, log rank test) and, the 1-year kidney graft survival was 100% in the TEG group vs 92.15% in the CCT group (P = 0.23, log tank test). CONCLUSION: TEG is a promising tool in guiding judicious use of anticoagulation with concomitant prevention of graft loss due to thrombosis, and reduces the length of hospital stay.

Timing of Ureteric Stent Removal and Occurrence of Urological Complications after Kidney Transplantation: A Systematic Review and Meta-Analysis.

Implanting a ureteric stent during ureteroneocystostomy reduces the risk of leakage and ureteral stenosis after kidney transplantation (KTx), but it may also predispose to urinary tract infections (UTIs). The aim of this study is to determine the optimal timing for ureteric stent removal after KTx. Searches were performed in EMBASE, MEDLINE Ovid, Cochrane CENTRAL, Web of Science, and Google Scholar (until November 2017). For this systematic review, all aspects of the Cochrane Handbook for Interventional Systematic Reviews were followed and it was written based on the PRISMA-statement. Articles discussing JJ-stents (double-J stents) and their time of removal in relation to outcomes, UTIs, urinary leakage, ureteral stenosis or reintervention were included. One-thousand-and-forty-three articles were identified, of which fourteen articles (three randomised controlled trials, nine retrospective cohort studies, and two prospective cohort studies) were included (describing in total n = 3612 patients). Meta-analysis using random effect models showed a significant reduction of UTIs when stents were removed earlier than three weeks (OR 0.49, CI 95%, 0.33 to 0.75, p = 0.0009). Regarding incidence of urinary leakage, there was no significant difference between early (<3 weeks) and late stent removal (>3 weeks) (OR 0.60, CI 95%, 0.29 to 1.23, p = 0.16). Based on our results, earlier stent removal (<3 weeks) was associated with a decreased incidence of UTIs and did not show a higher incidence of urinary leakage compared to later removal (>3 weeks). We recommend that the routine removal of ureteric stents implanted during KTx should be performed around three weeks post-operatively.