HygY Is a Twitch Radical SAM Epimerase with Latent Dehydrogenase Activity Revealed upon Mutation of a Single Cysteine Residue.

HygY is a SPASM/twitch radical SAM enzyme hypothesized to catalyze the C2'-epimerization of galacamine during the biosynthesis of hygromycin B. This activity is confirmed via biochemical and structural analysis of the derivatized reaction products using chemically synthesized deuterated substrate, high-resolution mass spectrometry and 1H NMR. Electron paramagnetic resonance spectroscopy of the reduced enzyme is consistent with ligation of two [Fe4S4] clusters characteristic of the twitch radical SAM subgroup. HygY catalyzed epimerization proceeds with incorporation of a single solvent Hydron into the talamine product facilitated by the catalytic cysteine-183 residue. Mutation of this cysteine to alanine converts HygY from a C2'-epimerase to an C2'-dehydrogenase with comparable activity. The SPASM/twitch radical SAM enzymes often serve as anaerobic oxidases making the redox-neutral epimerases in this class rather interesting. The discovery of latent dehydrogenase activity in a twitch epimerase may therefore offer new insights into the mechanistic features that distinguish oxidative versus redox-neutral SPASM/twitch enzymes and lead to the evolution of new enzyme activities.

Rapid in vitro assays for screening neutralizing antibodies and antivirals against SARS-CoV-2

Towards the end of 2019, a novel coronavirus (CoV) named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), genetically similar to severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1), emerged in Wuhan, Hubei province of China, and has been responsible of coronavirus disease 2019 (COVID-19) in humans. Since its first report, SARS-CoV-2 has resulted in a global pandemic, with over 10 million human infections and over 560,000 deaths reported worldwide at the end of June 2020. Currently, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines and/or antivirals licensed against SARS-CoV-2, and the high economical and health impact of SARS-CoV-2 has placed global pressure on the scientific community to identify effective prophylactic and therapeutic treatments for the treatment of SARS-CoV-2 infection and associated COVID-19 disease. While some compounds have been already reported to reduce SARS-CoV-2 infection and a handful of monoclonal antibodies (mAbs) have been described that neutralize SARS-CoV-2, there is an urgent need for the development and standardization of assays which can be used in high through-put screening (HTS) settings to identify new antivirals and/or neutralizing mAbs against SARS-CoV-2. Here, we described a rapid, accurate and highly reproducible plaque reduction microneutralization (PRMNT) assay that can be quickly adapted for the identification and characterization of both neutralizing mAbs and antivirals against SARS-CoV-2. Importantly, our MNA is compatible with HTS settings to interrogate large and/or complex libraries of mAbs and/or antivirals to identify those with neutralizing and/or antiviral activity, respectively, against SARS-CoV-2.

RNA-Independent DNA Cleavage Activities of Cas9 and Cas12a.

CRISPR-Cas systems provide bacteria and archaea with sequence-specific protection against invading mobile genetic elements. In the presence of divalent metal ions, Cas9 and Cas12a (formerly Cpf1) proteins target and cleave DNA that is complementary to a cognate guide RNA. The recognition of a protospacer adjacent motif (PAM) sequence in the target DNA by Cas9 and Cas12a is essential for cleavage. This RNA-guided DNA targeting is widely used for gene-editing methods. Here, we show that Francisella tularensis novicida (Fno) Cas12a, FnoCas9, and Streptococcus pyogenes Cas9 (SpyCas9) cleave DNA without a guide RNA in the presence of Mn2+ ions. Substrate requirements for the RNA-independent activity vary. FnoCas9 preferentially nicks double-stranded plasmid, SpyCas9 degrades single-stranded plasmid, and FnoCas12a cleaves both substrates. These observations suggest that the identities and levels of intracellular metals, along with the Cas9/Cas12a ortholog employed, could have significant impacts in genome editing applications.

Resolution of biliary stricture after living donor liver transplantation in a child by percutaneous trans-hepatic cholangiography and drainage: a case report.

INTRODUCTION: Intra-hepatic cholestasis arising from biliary strictures is a frequent complication in pediatric patients after liver transplantation. Minimally invasive procedures such as percutaneous drainage placement and balloon dilation are the preferred diagnostic and therapeutic modalities. CASE PRESENTATION: We report the case of a 12-month-old Caucasian boy with biliary atresia who was initially treated with hepatoportoenterostomy. In the following months, he developed biliary cirrhosis, accompanied by cystic bile retention, recurrent bile duct infections and malabsorption. Six months after the initial surgical intervention, he underwent living donor liver transplantation. Within two months, the hepatico-jejunostomy became occluded leading to progressive intra-hepatic cholestasis. Under sonographic guidance, external drainage of bile was accomplished by percutaneous trans-hepatic cholangiography and drainage. In total, our patient underwent 12 interventions under general anesthesia until balloon dilatation of the hepatico-jejunostomy was successfully performed. Finally, our patient's general condition improved and he gained weight. CONCLUSIONS: Minimally invasive techniques are preferred to surgical revisions and justify even multiple attempts. Interventions under general anesthesia, though not without risks, are still reasonable. Co-operation with parents and multidisciplinary approach to complication management by the involved surgeon, radiologist, pediatrician and anesthesiologist are important.

Evolution of pancreas transplantation: long-term results and perspectives from a high-volume center.

OBJECTIVE: To describe the evolution of pancreas transplantation from 1979 to 2011. The aim was to examine factors influencing long-term patient and graft survival, surgical methods, and risk factors influencing organ performance after transplantation. BACKGROUND: Pancreas transplantation has become the therapy of choice for patients suffering insulin-dependent diabetes and end stage renal failure. METHODS: Retrospective analysis of 509 consecutive pancreas transplants (442 simultaneous pancreas and kidney [SPK], 20 pancreas transplanted alone [PTA], and 47 pancreas transplanted after kidney [PAK]), performed at the University Hospital Innsbruck. The data were statistically analyzed using the Kaplan-Meier method and log-rank test. RESULTS: After overcoming initial immunological and technical problems between 1979 and 1988 (5-year pancreas graft survival rate, 29.7%), pancreas transplantation evolved during the second decade (1989-1996; 5-year pancreas graft survival rate, 42.2%). Technical changes, optimized immunosuppression, careful pretransplant evaluation, and improved graft monitoring have become standard in the last decade and result in excellent 5-year patient (94.3%), kidney (89.4%), and pancreas (81.5%) graft survival. Five-year graft survival was superior in SPK (68.8%) compared with PAK (62.5%) and PTA (16.4%). SPK retransplantation can be carried out safely with 5-year patient (87.5%) and pancreas graft (75.0%) survival. Overall 5-year patient survival after loss of the first pancreas graft is significantly better in patients who underwent retransplantation (89.4% vs. 67.9%, P = 0.001). Long-term pancreas graft survival is independent of donor body mass index, sex, and cause of death, anastomosis time and the number of human leukocyte antigen (HLA) mismatches, recipient age, body mass index, sex, current panel reactive antibodies, and waiting time. Significant risk factors for reduced graft survival are cold ischemia time and donor age. CONCLUSIONS: During the last 32 years, many problems in pancreas transplantation have been overcome and it may currently represent the therapeutic gold standard for some patients with diabetes and end stage renal failure.

Early viral load and recipient interleukin-28B rs12979860 genotype are predictors of the progression of hepatitis C after liver transplantation.

There have been few detailed studies of viral kinetics after liver transplantation (LT), and conflicting data have been reported on viral loads and the severity of recurrent hepatitis C virus (HCV) disease. This long-term study aimed to examine (1) the impact of HCV RNA levels at specific points in time within the first year and (2) the influence of interleukin-28B (IL-28B) genotypes on patient outcomes and the severity of recurrent HCV disease. The viral loads were measured 2, 4, 12, 24, and 48 weeks after LT, and the recipient/donor IL-28B genotypes of 164 patients were determined. A Cox regression analysis showed that the viral load at week 2 was an independent negative predictor of recipient outcomes. A week 2 viral load ≥ 6.0 log(10) IU/mL was significantly associated with reduced patient survival. After a mean follow-up of 6.5 years, 21 of 164 patients (12.8%) developed a cholestatic type of HCV recurrence and/or rapidly progressed to cirrhosis within 1 year. A multivariate binary regression analysis showed that HCV viremia at week 2 and a non-C/C recipient IL-28B genotype were independent risk factors for cholestatic recurrent HCV. No predictive factors could be found for the occurrence of recurrent liver cirrhosis 5 and 10 years after LT. Our study shows that the HCV RNA level at week 2 and the recipient IL-28B genotype are independent, statistically significant risk factors for post-LT cholestatic HCV, and it emphasizes the importance of viral load monitoring and IL-28B genotyping for identifying HCV recipients at risk for severe HCV recurrence.

Enteroscopic biopsies in the management of pancreas transplants: a proof of concept study for a novel monitoring tool.

BACKGROUND: Although percutaneous biopsies are considered to be the gold standard in diagnosing pancreas graft rejection, they are not performed routinely because of their association with severe complications. On the other hand, correct diagnosis of rejection is essential but may be difficult in cases of enteric drainage, particularly in patients with a pancreas transplant alone or a pancreas after kidney transplant. METHODS: Pancreas recipients who underwent enteroscopy between May 2005 and September 2009 were included in this retrospective analysis. Biopsies were graded 0 to 4 for interstitial and vascular changes. RESULTS: During the study period a total of 65 simultaneous pancreas-kidney transplants, 13 pancreas after kidney transplants and 4 pancreas transplants alone were performed. Sixty-three patients underwent a single enteroscopy, 10 had two, and 6 had three or more. Indications were protocol graft monitoring (n=73), graft dysfunction (n=17), enteric hemorrhage (n=9), or other (n=3). The duodenal segment was accessed in 76 instances (75%) with abnormal findings in 23. A total of 69 biopsies were obtained and revealed normal mucosa in 49 cases (71%). Histology showed signs of acute rejection in 11 cases. The upper gastrointestinal tract was also assessed, and, in 13 cases, additional pathologies were identified including gastroduodenitis (n=10), gastric/duodenal ulcer (n=2), and hemorrhagic esophagitis (n=1). No procedure-related complication occurred. CONCLUSIONS: This series of enteroscopies demonstrates that the duodenal segment of a pancreatic graft is accessible using our implant technique, and thus permitting biopsies to be obtained and endoscopic interventions to be performed.

Single-center experience with third and fourth kidney transplants.

Kidney retransplantation is often associated with a higher immunological risk than is primary renal transplantation. Faced with increasing organ shortage and growing waiting lists, results of kidney retransplantation are of particular interest. Fifty-six third and fourth kidney transplants were analyzed retrospectively. Parameters included patient and donor demographics, operative details, incidence of surgical, immunological and infectious complications and patient and graft survival. Patients receiving third kidney grafts had 1- and 5-year patient/graft survival rates of 97.4%/72.9% and 88.9%/53.6%, respectively. Episodes of acute rejection and delayed graft function were observed in 44% and 49% of these patients. Fourth kidney transplantation was associated with 1- and 2-year patient/graft survival rates of 84.8%/68.5% and 63.6%/47%, respectively. Acute rejection and delayed graft function occurred in 33% and in 60% of cases. Acceptable patient and graft survival may be achieved after third and fourth kidney transplantation. Graft losses in this sensitized population are mainly because of rejection. Profound immunosuppression may lead to major infectious problems.

Cytomegalovirus mismatch as major risk factor for delayed graft function after pancreas transplantation.

BACKGROUND: Risk factors for delayed graft function (DGF) in pancreas transplantation (PTx) and its implications on graft survival are poorly defined. METHODS: Eighty-seven consecutive first-time PTx for type I diabetes performed between January 2003 and December 2007 were retrospectively reviewed. DGF was defined as a reversible need for exogenous insulin beyond postoperative day 10 (DGF group [DGFG]). For statistical analysis, DGFG patients were compared with patients with immediate graft function (control group [CG]). RESULTS: DGF occurred in 16 patients (18.6%). C-peptide levels and DGF were inversely correlated (r=0.24, P=0.03). In univariate analysis, donor cytomegalovirus (CMV)+ antibody status, and D+/R- CMV mismatch were significantly associated with DGF (81.3% vs. CG 52.1%, P=0.029; and 62.5% vs. CG 21.1%, P=0.002, respectively). Compared with University of Wisconsin solution, histidine tryptophan ketoglutarate-preserved grafts displayed higher DGF rates (37.5% vs. CG 12.7%, P=0.030), similar to female recipients (DGFG 68.8% vs. CG 35.2%, P=0.015). On multivariate analysis, a significantly higher DGF incidence was noted in female recipients (DGFG 68.8% vs. CG 35.2%; P=0.03) and in recipients with D+/R- CMV mismatch (DGFG 62.5% vs. CG 21.1%; P=0.03). With a median follow-up of 40.4 months (range 0.7-74.2), graft survival at 5 years did not differ between both groups (94.4% CG vs. 93.8% DGFG; P=0.791). CONCLUSION: This is the first study that identifies CMV mismatch (D+/R-) as an additional risk factor for DGF occurrence in PTx. In this particular cohort, DGF does not seem to affect graft survival.

Cold ischemia contributes to the development of chronic rejection and mitochondrial injury after cardiac transplantation.

Chronic rejection (CR) remains an unsolved hurdle for long-term heart transplant survival. The effect of cold ischemia (CI) on progression of CR and the mechanisms resulting in functional deficit were investigated by studying gene expression, mitochondrial function, and enzymatic activity. Allogeneic (Lew→F344) and syngeneic (Lew→Lew) heart transplantations were performed with or without 10 h of CI. After evaluation of myocardial contraction, hearts were excised at 2, 10, 40, and 60 days for investigation of vasculopathy, gene expression, enzymatic activities, and mitochondrial respiration. Gene expression studies identified a gene cluster coding for subunits of the mitochondrial electron transport chain regulated in response to CI and CR. Myocardial performance, mitochondrial function, and mitochondrial marker enzyme activities declined in all allografts with time after transplantation. These declines were more rapid and severe in CI allografts (CR-CI) and correlated well with progression of vasculopathy and fibrosis. Mitochondria related gene expression and mitochondrial function are substantially compromised with the progression of CR and show that CI impacts on progression, gene profile, and mitochondrial function of CR. Monitoring mitochondrial function and enzyme activity might allow for earlier detection of CR and cardiac allograft dysfunction.

Extensive surveillance promotes early diagnosis and improved survival of de novo malignancies in liver transplant recipients.

The aim of our study was to examine whether an extensive surveillance protocol will promote early diagnosis and improved survival in patients with de novo cancer following liver transplantation (LT). Of 779 consecutive LT recipients, 96 (12.3%) developed 105 malignancies. The cumulative risk for the development of de novo cancer was 10%, 24%, 32% and 42% at 5, 10, 15 and 20 years after LT, respectively. The most frequent tumor types were skin (17%), lung (16%), oropharyngeal (11%) and prostate cancer (11%). The overall standard incidence ratio as compared to that of the general population was 1.9 (95% CI: 1.5-2.3). The median survival of patients with de novo non-skin cancers was 3.1 years after diagnosis. Only patients with skin cancers and solid tumors, diagnosed at early stages, showed an excellent outcome. After introducing an intensified surveillance protocol, the detection rate of de novo cancers increased from 4.9% to 13% and more de novo malignancies were diagnosed in earlier stages. For non-skin cancers, the median tumor-related survival significantly improved from 1.2 to 3.3 years as well as the median overall survival post-LT. This study indicates that an extensive tumor surveillance program is highly recommendable in LT recipients.

Transjugular intrahepatic portosystemic shunt in liver transplant recipients.

AIM: To evaluate the efficacy of transjugular intrahepatic portosystemic shunts (TIPSs) after liver transplantation (LT). METHODS: Between November 1996 and December 2005, 10 patients with severe recurrent hepatitis C virus infection (n = 4), ductopenic rejection (n = 5) or portal vein thrombosis (n = 1) were included in this analysis. Eleven TIPSs (one patient underwent two TIPS procedures) were placed for management of therapy-refractory ascites (n = 7), hydrothorax (n = 2) or bleeding from colonic varices (n = 1). The median time interval between LT and TIPS placement was 15 (4-158) mo. RESULTS: TIPS placement was successful in all patients. The mean portosystemic pressure gradient was reduced from 12.5 to 8.7 mmHg. Complete and partial remission could be achieved in 43% and 29% of patients with ascites. Both patients with hydrothorax did not respond to TIPS. No recurrent bleeding was seen in the patient with colonic varices. Nine of 10 patients died during the study period. Only one of two patients, who underwent retransplantation after the TIPS procedure, survived. The median survival period after TIPS placement was 3.3 (range 0.4-20) mo. The majority of patients died from sepsis with multiorgan failure. CONCLUSION: Indications for TIPS and technical performance in LT patients correspond to those in non-transplanted patients. At least partial control of therapy-refractory ascites and variceal bleeding could be achieved in most patients. Nevertheless, survival rates were disappointing, most probably because of the advanced stages of liver disease at the time of TIPS placement and the high risk of sepsis as a consequence of immunosuppression.

Fever of unknown origin in renal transplant patients with tacrolimus.

The immunosuppressive agent tacrolimus is now widely used for the prevention of acute and chronic rejection in renal allograft recipients. We here report on three patients, who developed drug-induced fever due to tacrolimus one to five months after renal transplantation. Extensive search for a focus, autoantibodies and virus infection remained inconclusive. Therefore, drug-induced fever was suggested. After discontinuing tacrolimus and switching to cyclosporine A fever completely resolved within 24 h. This report demonstrates that tacrolimus-induced drug fever should be included in the differential diagnosis of fever of unknown origin in renal transplant recipients.

A prospective randomized multicenter trial comparing histidine-tryptophane-ketoglutarate versus University of Wisconsin perfusion solution in clinical pancreas transplantation.

We aimed to evaluate early pancreas transplant graft function after histidine-tryptophan-ketoglutarate (HTK) versus University of Wisconsin (UW) perfusion. Prospective randomized multicenter study including 68 pancreas transplantations stratified according to preservation fluid used (27 HTK vs. 41 UW). Primary endpoint was pancreas graft survival at 6 months. Serum alpha-amylase, lipase, C-peptide, HbA1C and exogenous insulin requirement were compared at several time points. Mean pancreas cold ischemia time was 10.8 +/- 3.7 (HTK) vs. 11.8 +/- 3.4 h (UW) (P = 0.247). Simultaneous pancreas-kidney transplantation was performed in 95.6% of the patients, pancreas transplantation alone in 2.9%, and pancreas after kidney transplantation in 1.5%. Six months graft survival was 85.2% (HTK) vs. 90.2% (UW) (P = 0.703). Serum amylase and lipase values did not differ between both the groups during the observation period. C-peptide levels were elevated in both the groups without significant differences at each time point. Higher exogenous insulin requirement early after transplantation in the UW group had resolved at 3 months. Six month patient survival was 96.3% (HTK) vs. 100% (UW) (P = 0.397). With a mean cold ischemia time of 10 h in this study, HTK and UW solutions appear to be equally suitable for perfusion and organ preservation in clinical pancreas transplantation.

Early detection of emerging forest disease using dispersal estimation and ecological niche modeling.

Distinguishing the manner in which dispersal limitation and niche requirements control the spread of invasive pathogens is important for prediction and early detection of disease outbreaks. Here, we use niche modeling augmented by dispersal estimation to examine the degree to which local habitat conditions vs. force of infection predict invasion of Phytophthora ramorum, the causal agent of the emerging infectious tree disease sudden oak death. We sampled 890 field plots for the presence of P. ramorum over a three-year period (2003-2005) across a range of host and abiotic conditions with variable proximities to known infections in California, USA. We developed and validated generalized linear models of invasion probability to analyze the relative predictive power of 12 niche variables and a negative exponential dispersal kernel estimated by likelihood profiling. Models were developed incrementally each year (2003, 2003-2004, 2003-2005) to examine annual variability in model parameters and to create realistic scenarios for using models to predict future infections and to guide early-detection sampling. Overall, 78 new infections were observed up to 33.5 km from the nearest known site of infection, with slightly increasing rates of prevalence across time windows (2003, 6.5%; 2003-2004, 7.1%; 2003-2005, 9.6%). The pathogen was not detected in many field plots that contained susceptible host vegetation. The generalized linear modeling indicated that the probability of invasion is limited by both dispersal and niche constraints. Probability of invasion was positively related to precipitation and temperature in the wet season and the presence of the inoculum-producing foliar host Umbellularia californica and decreased exponentially with distance to inoculum sources. Models that incorporated niche and dispersal parameters best predicted the locations of new infections, with accuracies ranging from 0.86 to 0.90, suggesting that the modeling approach can be used to forecast locations of disease spread. Application of the combined niche plus dispersal models in a geographic information system predicted the presence of P. ramorum across approximately 8228 km2 of California's 84785 km2 (9.7%) of land area with susceptible host species. This research illustrates how probabilistic modeling can be used to analyze the relative roles of niche and dispersal limitation in controlling the distribution of invasive pathogens.

Markers of cellular senescence in zero hour biopsies predict outcome in renal transplantation.

Although chronological donor age is the most potent predictor of long-term outcome after renal transplantation, it does not incorporate individual differences of the aging-process itself. We therefore hypothesized that an estimate of biological organ age as derived from markers of cellular senescence in zero hour biopsies would be of higher predictive value. Telomere length and mRNA expression levels of the cell cycle inhibitors CDKN2A (p16INK4a) and CDKN1A (p21WAF1) were assessed in pre-implantation biopsies of 54 patients and the association of these and various other clinical parameters with serum creatinine after 1 year was determined. In a linear regression analysis, CDKN2A turned out to be the best single predictor followed by donor age and telomere length. A multiple linear regression analysis revealed that the combination of CDKN2A values and donor age yielded even higher predictive values for serum creatinine 1 year after transplantation. We conclude that the molecular aging marker CDKN2A in combination with chronological donor age predict renal allograft function after 1 year significantly better than chronological donor age alone.

Mitochondrial ischemia-reperfusion injury of the transplanted rat heart: improved protection by preservation versus cardioplegic solutions.

Cold ischemia time and preservation of organs are limited by I/R injury leading to primary nonfunction of the graft. In a rat heart transplant model, we compared cardioplegic St Thomas (ST) to histidine-tryptophan-ketoglutarate (HTK) and University of Wisconsin preservation solutions in terms of contractile function, and mitochondrial respiratory and enzymatic defects after prolonged cold ischemia and reperfusion. Contractile function was scored after transplantation and 24 h of reperfusion. Mitochondrial function was investigated by high-resolution respirometry of permeabilized myocardial fibers. Graft performance in terms of contractile function declined with the duration of cold storage. Recovery was significantly improved after 10 h of cold storage in HTK compared with ST (cardiac scores, 3.3+/-0.5 and 1.8+/-0.8, respectively). Tissue lactate dehydrogenase was better preserved in HTK than ST. Increase of tissue water content (edema) was less pronounced in HTK than ST (3.33+/-0.14 and 3.73+/-0.21 mg/mg dry weight, respectively). Similar cardiac scores (2.6+/-0.9 and 2.9+/-1.2, respectively) and mitochondrial respiratory parameters were obtained after preservation in HTK and University of Wisconsin. Decline in contractile function of individual grafts correlated well with loss of mitochondrial respiratory capacity, whereas citrate synthase activity remained largely preserved, indicating specific damage of respiratory complexes. Our data provide evidence for the superiority of preservation solutions versus a cardioplegic solution for prolonged cold storage of the heart. The correlation of graft performance and mitochondrial function indicates the potential of high-resolution respirometry for quantitative assessment of myocardial injury upon cold I/R, providing a basis for diagnostic approaches and evaluation of improved preservation solutions for heart transplantation.