Structural and mechanistic basis of the central energy-converting methyltransferase complex of methanogenesis.

Methanogenic archaea inhabiting anaerobic environments play a crucial role in the global biogeochemical material cycle. The most universal electrogenic reaction of their methane-producing energy metabolism is catalyzed by N    5-methyl-tetrahydromethanopterin: coenzyme M methyltransferase (MtrABCDEFGH), which couples the vectorial Na+ transport with a methyl transfer between the one-carbon carriers tetrahydromethanopterin and coenzyme M via a vitamin B12 derivative (cobamide) as prosthetic group. We present the 2.08 Šcryo-EM structure of Mtr(ABCDEFG)3 composed of the central Mtr(ABFG)3 stalk symmetrically flanked by three membrane-spanning MtrCDE globes. Tetraether glycolipids visible in the map fill gaps inside the multisubunit complex. Putative coenzyme M and Na+ were identified inside or in a side-pocket of a cytoplasmic cavity formed within MtrCDE. Its bottom marks the gate of the transmembrane pore occluded in the cryo-EM map. By integrating Alphafold2 information, functionally competent MtrA-MtrH and MtrA-MtrCDE subcomplexes could be modeled and thus the methyl-tetrahydromethanopterin demethylation and coenzyme M methylation half-reactions structurally described. Methyl-transfer-driven Na+ transport is proposed to be based on a strong and weak complex between MtrCDE and MtrA carrying vitamin B12, the latter being placed at the entrance of the cytoplasmic MtrCDE cavity. Hypothetically, strongly attached methyl-cob(III)amide (His-on) carrying MtrA induces an inward-facing conformation, Na+ flux into the membrane protein center and finally coenzyme M methylation while the generated loosely attached (or detached) MtrA carrying cob(I)amide (His-off) induces an outward-facing conformation and an extracellular Na+ outflux. Methyl-cob(III)amide (His-on) is regenerated in the distant active site of the methyl-tetrahydromethanopterin binding MtrH implicating a large-scale shuttling movement of the vitamin B12-carrying domain.

Differences in regulation mechanisms of glutamine synthetases from methanogenic archaea unveiled by structural investigations.

Glutamine synthetases (GS) catalyze the ATP-dependent ammonium assimilation, the initial step of nitrogen acquisition that must be under tight control to fit cellular needs. While their catalytic mechanisms and regulations are well-characterized in bacteria and eukaryotes, only limited knowledge exists in archaea. Here, we solved two archaeal GS structures and unveiled unexpected differences in their regulatory mechanisms. GS from Methanothermococcus thermolithotrophicus is inactive in its resting state and switched on by 2-oxoglutarate, a sensor of cellular nitrogen deficiency. The enzyme activation overlays remarkably well with the reported cellular concentration for 2-oxoglutarate. Its binding to an allosteric pocket reconfigures the active site through long-range conformational changes. The homolog from Methermicoccus shengliensis does not harbor the 2-oxoglutarate binding motif and, consequently, is 2-oxoglutarate insensitive. Instead, it is directly feedback-inhibited through glutamine recognition by the catalytic Asp50'-loop, a mechanism common to bacterial homologs, but absent in M. thermolithotrophicus due to residue substitution. Analyses of residue conservation in archaeal GS suggest that both regulations are widespread and not mutually exclusive. While the effectors and their binding sites are surprisingly different, the molecular mechanisms underlying their mode of action on GS activity operate on the same molecular determinants in the active site.

The Mononuclear Metal-Binding Site of Mo-Nitrogenase Is Not Required for Activity.

The biological N2-fixation process is catalyzed exclusively by metallocofactor-containing nitrogenases. Structural and spectroscopic studies highlighted the presence of an additional mononuclear metal-binding (MMB) site, which can coordinate Fe in addition to the two metallocofactors required for the reaction. This MMB site is located 15-Å from the active site, at the interface of two NifK subunits. The enigmatic function of the MMB site and its implications for metallocofactor installation, catalysis, electron transfer, or structural stability are investigated in this work. The axial ligands coordinating the additional Fe are almost universally conserved in Mo-nitrogenases, but a detailed observation of the available structures indicates a variation in occupancy or a metal substitution. A nitrogenase variant in which the MMB is disrupted was generated and characterized by X-ray crystallography, biochemistry, and enzymology. The crystal structure refined to 1.55-Å revealed an unambiguous loss of the metal site, also confirmed by an absence of anomalous signal for Fe. The position of the surrounding side chains and the overall architecture are superposable with the wild-type structure. Accordingly, the biochemical and enzymatic properties of the variant are similar to those of the wild-type nitrogenase, indicating that the MMB does not impact nitrogenase's activity and stability in vitro.

Sensitive and selective phenol sensing in denitrifying Aromatoleum aromaticum EbN1T.

IMPORTANCE: Aromatic compounds are globally abundant organic molecules with a multitude of natural and anthropogenic sources, underpinning the relevance of their biodegradation. A. aromaticum EbN1T is a well-studied environmental betaproteobacterium specialized on the anaerobic degradation of aromatic compounds. The here studied responsiveness toward phenol in conjunction with the apparent high ligand selectivity (non-promiscuity) of its PheR sensor and those of the related p-cresol (PcrS) and p-ethylphenol (EtpR) sensors are in accord with the substrate-specificity and biochemical distinctiveness of the associated degradation pathways. Furthermore, the present findings advance our general understanding of the substrate-specific regulation of the strain's remarkable degradation network and of the concentration thresholds below which phenolic compounds become essentially undetectable and as a consequence should escape substantial biodegradation. Furthermore, the findings may inspire biomimetic sensor designs for detecting and quantifying phenolic contaminants in wastewater or environments.

Bioelectrocatalytic CO2 Reduction by Mo-Dependent Formylmethanofuran Dehydrogenase.

Massive efforts are invested in developing innovative CO2 -sequestration strategies to counter climate change and transform CO2 into higher-value products. CO2 -capture by reduction is a chemical challenge, and attention is turned toward biological systems that selectively and efficiently catalyse this reaction under mild conditions and in aqueous solvents. While a few reports have evaluated the effectiveness of isolated bacterial formate dehydrogenases as catalysts for the reversible electrochemical reduction of CO2 , it is imperative to explore other enzymes among the natural reservoir of potential models that might exhibit higher turnover rates or preferential directionality for the reductive reaction. Here, we present electroenzymatic catalysis of formylmethanofuran dehydrogenase, a CO2 -reducing-and-fixing biomachinery isolated from a thermophilic methanogen, which was deposited on a graphite rod electrode to enable direct electron transfer for electroenzymatic CO2 reduction. The gas is reduced with a high Faradaic efficiency (109±1 %), where a low affinity for formate prevents its electrochemical reoxidation and favours formate accumulation. These properties make the enzyme an excellent tool for electroenzymatic CO2 -fixation and inspiration for protein engineering that would be beneficial for biotechnological purposes to convert the greenhouse gas into stable formate that can subsequently be safely stored, transported, and used for power generation without energy loss.

[Surgical treatment of pseudomyxoma peritonei-Cytoreductive surgery and hyperthermic intraperitoneal chemotherapy]. / Chirurgische Therapie des Pseudomyxoma-peritonei-Syndroms ­ zytoreduktive Chirurgie und hypertherme intraperitoneale Chemotherapie.

BACKGROUND: Pseudomyxoma peritonei syndrome (PMP) is an orphan disease. Surgery is the fundament of treatment. METHOD: Short review summarizing the state of the art treatment. RESULTS: Cytoreductive surgery (CRS) in combination with hyperthermic intraperitoneal chemotherapy (HIPEC) form the foundations of treatment for PMP. The peritoneal cancer index should be preoperatively determined based on imaging and/or laparoscopy, intraoperatively validated and both should be documented. An extraperitoneal surgical preparation technique leads to effective en bloc resection of the peritoneum and the affected abdominal area. The HIPEC technique should be performed with mitomycin C for 60-90 min. Complete CRS (CC = 0, CC = 1) and the histological subtype are relevant for the prognosis. Structured educational programs and mentoring can optimize the learning curve. The aftercare should be performed at the surgical center. After follow-up imaging at 3 months after CRS, in the first 2 years a control should be carried out every 6 months. Thereafter, the intervals can be extended to 1 year. CONCLUSION: Standardized surgical treatment and HIPEC, optimized specific surgical training and structured follow-up at the center lead to an excellent long-term prognosis for patients with PMP.

Imaged guided surgery during arteriovenous malformation of gastrointestinal stromal tumor using hyperspectral and indocyanine green visualization techniques: A case report.

BACKGROUND: This case report demonstrates the simultaneous development of a gastrointestinal stromal tumour (GIST) with arteriovenous malformations (AVMs) within the jejunal mesentery. A 74-year-old male presented to the department of surgery at our institution with a one-month history of abdominal pain. Contrast-enhanced computed tomography revealed an AVM. During exploratory laparotomy, hyperspectral imaging (HSI) and indocyanine green (ICG) fluorescence were used to evaluate the extent of the tumour and determine the resection margins. Intraoperative imaging confirmed AVM, while histopathological evaluation showed an epithelioid, partially spindle cell GIST. CASE SUMMARY: This is the first case reporting the use of HSI and ICG to image GIST intermingled with an AVM. The resection margins were planned using intraoperative analysis of additional optical data. Image-guided surgery enhances the clinician's knowledge of tissue composition and facilitates tissue differentiation. CONCLUSION: Since image-guided surgery is safe, this procedure should increase in popularity among the next generation of surgeons as it is associated with better postoperative outcomes.

High resolution cryo-EM and crystallographic snapshots of the actinobacterial two-in-one 2-oxoglutarate dehydrogenase.

Actinobacteria possess unique ways to regulate the oxoglutarate metabolic node. Contrary to most organisms in which three enzymes compose the 2-oxoglutarate dehydrogenase complex (ODH), actinobacteria rely on a two-in-one protein (OdhA) in which both the oxidative decarboxylation and succinyl transferase steps are carried out by the same polypeptide. Here we describe high-resolution cryo-EM and crystallographic snapshots of representative enzymes from Mycobacterium smegmatis and Corynebacterium glutamicum, showing that OdhA is an 800-kDa homohexamer that assembles into a three-blade propeller shape. The obligate trimeric and dimeric states of the acyltransferase and dehydrogenase domains, respectively, are critical for maintaining the overall assembly, where both domains interact via subtle readjustments of their interfaces. Complexes obtained with substrate analogues, reaction products and allosteric regulators illustrate how these domains operate. Furthermore, we provide additional insights into the phosphorylation-dependent regulation of this enzymatic machinery by the signalling protein OdhI.

Assimilatory sulfate reduction in the marine methanogen Methanothermococcus thermolithotrophicus.

Methanothermococcus thermolithotrophicus is the only known methanogen that grows on sulfate as its sole sulfur source, uniquely uniting methanogenesis and sulfate reduction. Here we use physiological, biochemical and structural analyses to provide a snapshot of the complete sulfate reduction pathway of this methanogenic archaeon. We find that later steps in this pathway are catalysed by atypical enzymes. PAPS (3'-phosphoadenosine 5'-phosphosulfate) released by APS kinase is converted into sulfite and 3'-phosphoadenosine 5'-phosphate (PAP) by a PAPS reductase that is similar to the APS reductases of dissimilatory sulfate reduction. A non-canonical PAP phosphatase then hydrolyses PAP. Finally, the F420-dependent sulfite reductase converts sulfite to sulfide for cellular assimilation. While metagenomic and metatranscriptomic studies suggest that the sulfate reduction pathway is present in several methanogens, the sulfate assimilation pathway in M. thermolithotrophicus is distinct. We propose that this pathway was 'mix-and-matched' through the acquisition of assimilatory and dissimilatory enzymes from other microorganisms and then repurposed to fill a unique metabolic role.

Nitrogen Fixation and Hydrogen Evolution by Sterically Encumbered Mo-Nitrogenase.

The substrate-reducing proteins of all nitrogenases (MoFe, VFe, and FeFe) are organized as α2ß2(γ2) multimers with two functional halves. While their dimeric organization could afford improved structural stability of nitrogenases in vivo, previous research has proposed both negative and positive cooperativity contributions with respect to enzymatic activity. Here, a 1.4 kDa peptide was covalently introduced in the proximity of the P cluster, corresponding to the Fe protein docking position. The Strep-tag carried by the added peptide simultaneously sterically inhibits electron delivery to the MoFe protein and allows the isolation of partially inhibited MoFe proteins (where the half-inhibited MoFe protein was targeted). We confirm that the partially functional MoFe protein retains its ability to reduce N2 to NH3, with no significant difference in selectivity over obligatory/parasitic H2 formation. Our experiment concludes that wild-type nitrogenase exhibits negative cooperativity during the steady state regarding H2 and NH3 formation (under Ar or N2), with one-half of the MoFe protein inhibiting turnover in the second half. This emphasizes the presence and importance of long-range (>95 Å) protein-protein communication in biological N2 fixation in Azotobacter vinelandii.

Structural and biochemical elucidation of class I hybrid cluster protein natively extracted from a marine methanogenic archaeon.

Whilst widespread in the microbial world, the hybrid cluster protein (HCP) has been paradoxically a long-time riddle for microbiologists. During three decades, numerous studies on a few model organisms unravelled its structure and dissected its metal-containing catalyst, but the physiological function of the enzyme remained elusive. Recent studies on bacteria point towards a nitric oxide reductase activity involved in resistance during nitrate and nitrite reduction as well as host infection. In this study, we isolated and characterised a naturally highly produced HCP class I from a marine methanogenic archaeon grown on ammonia. The crystal structures of the enzyme in a reduced and partially oxidised state, obtained at a resolution of 1.45 and 1.36-Å, respectively, offered a precise picture of the archaeal enzyme intimacy. There are striking similarities with the well-studied enzymes from Desulfovibrio species regarding sequence, kinetic parameters, structure, catalyst conformations, and internal channelling systems. The close phylogenetic relationship between the enzymes from Methanococcales and many Bacteria corroborates this similarity. Indeed, Methanococcales HCPs are closer to these bacterial homologues than to any other archaeal enzymes. The relatively high constitutive production of HCP in M. thermolithotrophicus, in the absence of a notable nitric oxide source, questions the physiological function of the enzyme in these ancient anaerobes.

Structures of the sulfite detoxifying F420-dependent enzyme from Methanococcales.

Methanogenic archaea are main actors in the carbon cycle but are sensitive to reactive sulfite. Some methanogens use a sulfite detoxification system that combines an F420H2-oxidase with a sulfite reductase, both of which are proposed precursors of modern enzymes. Here, we present snapshots of this coupled system, named coenzyme F420-dependent sulfite reductase (Group I Fsr), obtained from two marine methanogens. Fsr organizes as a homotetramer, harboring an intertwined six-[4Fe-4S] cluster relay characterized by spectroscopy. The wire, spanning 5.4 nm, electronically connects the flavin to the siroheme center. Despite a structural architecture similar to dissimilatory sulfite reductases, Fsr shows a siroheme coordination and a reaction mechanism identical to assimilatory sulfite reductases. Accordingly, the reaction of Fsr is unidirectional, reducing sulfite or nitrite with F420H2. Our results provide structural insights into this unique fusion, in which a primitive sulfite reductase turns a poison into an elementary block of life.

Hyperspectral imaging detects perfusion and oxygenation differences between stapled and hand-sewn intestinal anastomoses.

Objectives: Hand-sewn and stapled intestinal anastomoses are both daily performed routine procedures by surgeons. Yet, differences in micro perfusion of these two surgical techniques and their impact on surgical outcomes are still insufficiently understood. Only recently, hyperspectral imaging (HSI) has been established as a non-invasive, contact-free, real-time assessment tool for tissue oxygenation and micro-perfusion. Hence, objective of this study was HSI assessment of different intestinal anastomotic techniques and analysis of patients' clinical outcome. Methods: Forty-six consecutive patients with an ileal-ileal anastomoses were included in our study; 21 side-to-side stapled and 25 end-to-end hand-sewn. Based on adsorption and reflectance of the analyzed tissue, chemical color imaging indicates oxygen saturation (StO2), tissue perfusion (near-infrared perfusion index [NIR]), organ hemoglobin index (OHI), and tissue water index (TWI). Results: StO2 as well as NIR of the region of interest (ROI) was significantly higher in stapled anastomoses as compared to hand-sewn ileal-ileal anastomoses (StO2 0.79 (0.74-0.81) vs. 0.66 (0.62-0.70); p<0.001 NIR 0.83 (0.70-0.86) vs. 0.70 (0.63-0.76); p=0.01). In both groups, neither anastomotic leakage nor abdominal septic complications nor patient death did occur. Conclusions: Intraoperative HSI assessment is able to detect significant differences in tissue oxygenation and NIR of hand-sewn and stapled intestinal anastomoses. Long-term clinical consequences resulting from the reduced tissue oxygenation and tissue perfusion in hand-sewn anastomoses need to be evaluated in larger clinical trials, as patients may benefit from further refined surgical techniques.

Hyperspectral Imaging for Viability Assessment of Human Liver Allografts During Normothermic Machine Perfusion.

Normothermic machine perfusion (NMP) is nowadays frequently utilized in liver transplantation. Despite commonly accepted viability assessment criteria, such as perfusate lactate and perfusate pH, there is a lack of predictive organ evaluation strategies to ensure graft viability. Hyperspectral imaging (HSI)-as an optical imaging modality increasingly applied in the biomedical field-might provide additional useful data regarding allograft viability and performance of liver grafts during NMP. Methods: Twenty-five deceased donor liver allografts were included in the study. During NMP, graft viability was assessed conventionally and by means of HSI. Images of liver parenchyma were acquired at 1, 2, and 4 h of NMP, and subsequently analyzed using a specialized HSI acquisition software to compute oxygen saturation, tissue hemoglobin index, near-infrared perfusion index, and tissue water index. To analyze the association between HSI parameters and perfusate lactate as well as perfusate pH, we performed simple linear regression analysis. Results: Perfusate lactate at 1, 2, and 4 h NMP was 1.5 [0.3-8.1], 0.9 [0.3-2.8], and 0.9 [0.1-2.2] mmol/L. Perfusate pH at 1, 2, and 4 h NMP was 7.329 [7.013-7.510], 7.318 [7.081-7.472], and 7.265 [6.967-7.462], respectively. Oxygen saturation predicted perfusate lactate at 1 and 2 h NMP (R2 = 0.1577, P = 0.0493; R2 = 0.1831, P = 0.0329; respectively). Tissue hemoglobin index predicted perfusate lactate at 1, 2, and 4 h NMP (R2 = 0.1916, P = 0.0286; R2 = 0.2900, P = 0.0055; R2 = 0.2453, P = 0.0139; respectively). Conclusions: HSI may serve as a noninvasive tool for viability assessment during NMP. Further evaluation and validation of HSI parameters are warranted in larger sample sizes.

Comparative Transcriptomics Sheds Light on Remodeling of Gene Expression during Diazotrophy in the Thermophilic Methanogen Methanothermococcus thermolithotrophicus.

Some marine thermophilic methanogens are able to perform energy-consuming nitrogen fixation despite deriving only little energy from hydrogenotrophic methanogenesis. We studied this process in Methanothermococcus thermolithotrophicus DSM 2095, a methanogenic archaeon of the order Methanococcales that contributes to the nitrogen pool in some marine environments. We successfully grew this archaeon under diazotrophic conditions in both batch and fermenter cultures, reaching the highest cell density reported so far. Diazotrophic growth depended strictly on molybdenum and, in contrast to other diazotrophs, was not inhibited by tungstate or vanadium. This suggests an elaborate control of metal uptake and a specific metal recognition system for the insertion into the nitrogenase cofactor. Differential transcriptomics of M. thermolithotrophicus grown under diazotrophic conditions with ammonium-fed cultures as controls revealed upregulation of the nitrogenase machinery, including chaperones, regulators, and molybdate importers, as well as simultaneous upregulation of an ammonium transporter and a putative pathway for nitrate and nitrite utilization. The organism thus employs multiple synergistic strategies for uptake of nitrogen nutrients during the early exponential growth phase without altering transcription levels for genes involved in methanogenesis. As a counterpart, genes coding for transcription and translation processes were downregulated, highlighting the maintenance of an intricate metabolic balance to deal with energy constraints and nutrient limitations imposed by diazotrophy. This switch in the metabolic balance included unexpected processes, such as upregulation of the CRISPR-Cas system, probably caused by drastic changes in transcription levels of putative mobile and virus-like elements. IMPORTANCE The thermophilic anaerobic archaeon M. thermolithotrophicus is a particularly suitable model organism to study the coupling of methanogenesis to diazotrophy. Likewise, its capability of simultaneously reducing N2 and CO2 into NH3 and CH4 with H2 makes it a viable target for biofuel production. We optimized M. thermolithotrophicus cultivation, resulting in considerably higher cell yields and enabling the successful establishment of N2-fixing bioreactors. Improved understanding of the N2 fixation process would provide novel insights into metabolic adaptations that allow this energy-limited extremophile to thrive under diazotrophy, for instance, by investigating its physiology and uncharacterized nitrogenase. We demonstrated that diazotrophic growth of M. thermolithotrophicus is exclusively dependent on molybdenum, and complementary transcriptomics corroborated the expression of the molybdenum nitrogenase system. Further analyses of differentially expressed genes during diazotrophy across three cultivation time points revealed insights into the response to nitrogen limitation and the coordination of core metabolic processes.

Ex situ arterial reconstruction prior normothermic machine perfusion of liver grafts.

PURPOSE: Atypical variants of the hepatic artery are common and pose a technical challenge for normothermic machine perfusion (NMP). The transplant surgeon has three options when confronted with hepatic arterial variation in a liver graft to be subjected to NMP: to perform arterial reconstruction (i) prior, (ii) during, or (iii) following NMP. METHODS: Herein, we report our experience and technical considerations with pre-NMP reconstruction. Out of 52 livers, 9 had an atypical hepatic artery (HA): 3 replaced right HA, 3 replaced left HA, 1 accessory left HA, 1 accessory left and right HA, and 1 replaced left and right HA. RESULTS: Reconstruction was conducted during back-table preparation. A single vascular conduit was created in all grafts to allow single arterial cannulation for NMP, necessitating only one arterial anastomosis within the recipient. All grafts were subjected to NMP and subsequently successfully transplanted. CONCLUSION: Our approach is being advocated for as it preserves the ability to alter the reconstruction in case of problems resulting from the reconstruction itself, thereby allowing functional evaluation of the reconstruction prior transplantation, permitting simultaneous reperfusion in the recipient, and providing the shortest possible duration for vascular reconstruction once the graft is rewarming non-perfused within the recipient. In addition, in light of the frequency of technically demanding reconstructions with very small vessels, we consider our technique beneficial as the procedure can be performed under ideal conditions at the back-table.

A Structural View of Alkyl-Coenzyme M Reductases, the First Step of Alkane Anaerobic Oxidation Catalyzed by Archaea.

Microbial anaerobic oxidation of alkanes intrigues the scientific community by way of its impact on the global carbon cycle, and its biotechnological applications. Archaea are proposed to degrade short- and long-chain alkanes to CO2 by reversing methanogenesis, a theoretically reversible process. The pathway would start with alkane activation, an endergonic step catalyzed by methyl-coenzyme M reductase (MCR) homologues that would generate alkyl-thiols carried by coenzyme M. While the methane-generating MCR found in methanogens has been well characterized, the enzymatic activity of the putative alkane-fixing counterparts has not been validated so far. Such an absence of biochemical investigations contrasts with the current explosion of metagenomics data, which draws new potential alkane-oxidizing pathways in various archaeal phyla. Therefore, validating the physiological function of these putative alkane-fixing machines and investigating how their structures, catalytic mechanisms, and cofactors vary depending on the targeted alkane have become urgent needs. The first structural insights into the methane- and ethane-capturing MCRs highlighted unsuspected differences and proposed some explanations for their substrate specificity. This Perspective reviews the current physiological, biochemical, and structural knowledge of alkyl-CoM reductases and offers fresh ideas about the expected mechanistic and chemical differences among members of this broad family. We conclude with the challenges of the investigation of these particular enzymes, which might one day generate biofuels for our modern society.

Hyperspectral Imaging for Assessment of Initial Graft Function in Human Kidney Transplantation.

The aim of our study was to evaluate hyperspectral imaging (HSI) as a rapid, non-ionizing technique for the assessment of organ quality and the prediction of delayed graft function (DGF) in kidney transplantation after static cold storage (SCS, n = 20), as well as hypothermic machine perfusion (HMP, n = 18). HSI assessment of the kidney parenchyma was performed during organ preservation and at 10 and 30 min after reperfusion using the TIVITA® Tissue System (Diaspective Vision GmbH, Am Salzhaff, Germany), calculating oxygen saturation (StO2), near-infrared perfusion index (NIR), tissue haemoglobin index (THI), and tissue water index (TWI). Recipient and donor characteristics were comparable between organ preservation groups. Cold ischemic time was significantly longer in the HMP group (14.1 h [3.6-23.1] vs. 8.7h [2.2-17.0], p = 0.002). The overall presence of DGF was comparable between groups (HMP group n = 10 (55.6%), SCS group n = 10 (50.0%)). Prediction of DGF was possible in SCS and HMP kidneys; StO2 at 10 (50.00 [17.75-76.25] vs. 63.17 [27.00-77.75]%, p = 0.0467) and 30 min (57.63 [18.25-78.25] vs. 65.38 [21.25-83.33]%, p = 0.0323) after reperfusion, as well as NIR at 10 (41.75 [1.0-58.00] vs. 48.63 [12.25-69.50], p = 0.0137) and 30 min (49.63 [8.50-66.75] vs. 55.80 [14.75-73.25], p = 0.0261) after reperfusion were significantly lower in DGF kidneys, independent of the organ preservation method. In conclusion, HSI is a reliable method for intraoperative assessment of renal microperfusion, applicable after organ preservation through SCS and HMP, and predicts the development of DGF.

Hyperspectral imaging for quantitative assessment of hepatic steatosis in human liver allografts.

INTRODUCTION: In liver transplantation (LT), steatosis is commonly judged to be a risk factor for graft dysfunction, and quantitative assessment of hepatic steatosis remains crucial. Liver biopsy as the gold standard for evaluation of hepatic steatosis has certain drawbacks, that is, invasiveness, and intra- and inter-observer variability. A non-invasive, quantitative modality could replace liver biopsy and eliminate these disadvantages, but has not yet been evaluated in human LT. METHODS: We performed a pilot study to evaluate the feasibility and accuracy of hyperspectral imaging (HSI) in the assessment of hepatic steatosis of human liver allografts for transplantation. Thirteen deceased donor liver allografts were included in the study. The degree of steatosis was assessed by means of conventional liver biopsy as well as HSI, performed at the end of back-table preparation, during normothermic machine perfusion (NMP), and after reperfusion in the recipient. RESULTS: Organ donors were 51 [30-83] years old, and 61.5% were male. Donor body mass index was 24.2 [16.5-38.0] kg/m2 . The tissue lipid index (TLI) generated by HSI at the end of back-table preparation correlated significantly with the histopathologically assessed degree of overall hepatic steatosis (R2 = .9085, P < .0001); this was based on a correlation of TLI and microvesicular steatosis (R2 = .8120; P < .0001). There is also a linear relationship between the histopathologically assessed degree of overall steatosis and TLI during NMP (R2 = .5646; P = .0031) as well as TLI after reperfusion (R2 = .6562; P = .0008). CONCLUSION: HSI may safely be applied for accurate assessment of hepatic steatosis in human liver grafts. Certainly, TLI needs further assessment and validation in larger sample sizes.