Efficient Transferase Engineering for SAM Analog Synthesis from Iodoalkanes.

S-Adenosyl-l-methionine (SAM) is an important cosubstrate in various biochemical processes, including selective methyl transfer reactions. Simple methods for the (re)generation of SAM analogs could expand the chemistry accessible with SAM-dependent transferases and go beyond methylation reactions. Here we present an efficient enzyme engineering strategy to synthesize different SAM analogs from "off-the-shelf" iodoalkanes through enzymatic alkylation of S-adenosyl-l-homocysteine (SAH). This was achieved by mutating multiple hydrophobic and structurally dynamic amino acids simultaneously. Combinatorial mutagenesis was guided by the natural amino acid diversity and generated a highly functional mutant library. This approach increased the speed as well as the scale of enzyme engineering by providing a panel of optimized enzymes with orders of magnitude higher activities for multiple substrates in just one round of enzyme engineering. The optimized enzymes exhibit catalytic efficiencies up to 31 M-1 s-1, convert various iodoalkanes, including substrates bearing cyclopropyl or aromatic moieties, and catalyze S-alkylation of SAH with very high stereoselectivities (>99 % de). We further report a high throughput chromatographic screening system for reliable and rapid SAM analog analysis. We believe that the methods and enzymes described herein will further advance the field of selective biocatalytic alkylation chemistry by enabling SAM analog regeneration with "off-the-shelf" reagents.

Selective Biocatalytic N-Methylation of Unsaturated Heterocycles.

Methods for regioselective N-methylation and -alkylation of unsaturated heterocycles with "off the shelf" reagents are highly sought-after. This reaction could drastically simplify synthesis of privileged bioactive molecules. Here we report engineered and natural methyltransferases for challenging N-(m)ethylation of heterocycles, including benzimidazoles, benzotriazoles, imidazoles and indazoles. The reactions are performed through a cyclic enzyme cascade that consists of two methyltransferases using only iodoalkanes or methyl tosylate as simple reagents. This method enables the selective synthesis of important molecules that are otherwise difficult to access, proceeds with high regioselectivity (r.r. up to >99 %), yield (up to 99 %), on a preparative scale, and with nearly equimolar concentrations of simple starting materials.

Hematological Manifestations among Patients with Rheumatic Diseases.

BACKGROUND: Rheumatic diseases have many hematological manifestations. Blood dyscrasias and other hematological abnormalities are sometimes the first sign of rheumatic disease. In addition, novel antirheumatic biological agents may cause cytopenias. SUMMARY: The aim of this review was to discuss cytopenias caused by systemic lupus erythematosus and antirheumatic drugs, Felty's syndrome in rheumatoid arthritis, and autoimmune hemolytic anemia, thrombosis, and thrombotic microangiopathies related to rheumatological conditions such as catastrophic antiphospholipid syndrome and scleroderma renal crisis. Key Message: The differential diagnosis of various hematological disorders should include rheumatic autoimmune diseases among other causes of blood cell and hemostasis abnormalities. It is crucial that hematologists be aware of these presentations so that they are diagnosed and treated in a timely manner.

Acute mesenteric ischemia (AMI): absence of renal insufficiency and performance of early bowel resection may indicate improved outcomes.

PURPOSE: Acute mesenteric ischemia (AMI) is still associated with very high morbidity and mortality while the rareness and heterogeneity hamper the establishment of evidence-based guidelines. We sought to help standardize contemporary treatment by a cohort study at our tertiary center in the rising endovascular age. METHODS: A retrospective cohort study was conducted from 2005 to 2015. Patients with occlusive (OMI), non-occlusive (NOMI), and venous mesenteric ischemia (VMI) were compared with respect to clinical and treatment parameters as well as outcome. RESULTS: The study cohort consisted of 48 patients composed of 27 males and 21 females with an average age of 63 years and an average BMI of 25.1 kg/m2. In 48% of patients (N=23), an acute arterial OMI had occurred while NOMI was present in 31% (N=15) and VMI in 21% (N=10). Interventional and intraoperative recanalizations were significantly more often required in OMI patients compared with other entities (p=0.003). Patients with venous mesenteric ischemia had a significant better overall survival than patients with OMI or NOMI in the univariate analysis (p=0.027). Patients with renal failure had a 14.7-fold higher relative risk (Cox p=0.013) and patients without bowel resection during primary surgery had a 17.8-fold higher relative risk (Cox p=0.047) to die of AMI in the postoperative course. CONCLUSIONS: AMI remains a rare but oftentimes fatal disease. Our study provides evidence that outcome may depend on the AMI subtype, presence of renal insufficiency, and early bowel resection. Further research should help individualize treatment for optimized outcomes.

Rheumatoid arthritis and lymphoma: Incidence, pathogenesis, biology, and outcome.

Patients with rheumatoid arthritis (RA) have a greater risk of developing both Hodgkin lymphoma (HL) and non-HL than the general population. Non-Hodgkin lymphoma is more common than HL in these patients, and diffuse large B cell lymphoma is the most frequent subtype observed. Although the clinical course of lymphoma in RA is often aggressive, the prognosis in these cases is similar to that of lymphoma in the general population. In this review, we summarize data derived from both retrospective and prospective studies, regarding incidence, pathogenesis, and outcome of lymphomas in RA patients and outline the possible mechanisms and hypotheses linking these 2 disorders. Over the years, 3 main theories have been suggested to explain this association. These hypotheses relate to genetic predisposition, persistence of long standing disease activity with continued immune stimulation, and the role of anti-RA therapy given. A common genetic predisposition linking RA and lymphoma has not been established. As for treatment of RA, this includes immunosuppressive antitumor necrosis factor drugs or conventional disease modifying antirheumatic drugs like methotrexate. Neither of these drug categories appears to be associated with a higher risk of lymphoma in RA. The impact of continuing disease activity and immune stimulation appears to be the most significant in lymphomagenesis in these patients.

Systemic lupus erythematosus and lymphoma: Incidence, pathogenesis and biology.

Systemic Lupus Erythematosus (SLE), a well recognized systemic autoimmune disease is associated with an increased risk of malignancies, particularly lymphoma. Various studies have shown this risk to be as high as 4-7-fold compared to the general population. The pathogenesis of lymphoma in patients with SLE is still not well understood. In this review we summarize the world literature and update current knowledge on the interesting link between SLE and lymphomagenesis. We relate in turn to incidence rates of lymphoma in SLE and subtypes of lymphoma encountered; pathogenesis and relevant theories proposed; links with EBV and the possible role of continued activity of lupus and of immunosuppressive therapy in lymphomagenesis. It is clearly evident that further studies are needed to improve the understanding of this association. Some cytokines and proteins associated with cell survival and proliferation, such as BAFF, APRIL, IL6 and BCL2, have been found to be elevated both in SLE and lymphoma. These factors may well impact pathogenesis, however, a direct "cause and effect" relationship is yet to be demonstrated.

Enhanced labeling density and whole-cell 3D dSTORM imaging by repetitive labeling of target proteins.

With continuing advances in the resolving power of super-resolution microscopy, the inefficient labeling of proteins with suitable fluorophores becomes a limiting factor. For example, the low labeling density achieved with antibodies or small molecule tags limits attempts to reveal local protein nano-architecture of cellular compartments. On the other hand, high laser intensities cause photobleaching within and nearby an imaged region, thereby further reducing labeling density and impairing multi-plane whole-cell 3D super-resolution imaging. Here, we show that both labeling density and photobleaching can be addressed by repetitive application of trisNTA-fluorophore conjugates reversibly binding to a histidine-tagged protein by a novel approach called single-epitope repetitive imaging (SERI). For single-plane super-resolution microscopy, we demonstrate that, after multiple rounds of labeling and imaging, the signal density is increased. Using the same approach of repetitive imaging, washing and re-labeling, we demonstrate whole-cell 3D super-resolution imaging compensated for photobleaching above or below the imaging plane. This proof-of-principle study demonstrates that repetitive labeling of histidine-tagged proteins provides a versatile solution to break the 'labeling barrier' and to bypass photobleaching in multi-plane, whole-cell 3D experiments.

Screening for occult cancer in idiopathic venous thromboembolism - Systemic review and meta-analysis.

BACKGROUND: Idiopathic venous thromboembolism (VTE) may be associated with an occult malignancy. Early detection of cancer might be translated to a better prognosis for these patients. However, the efficacy of extensive screening for cancer in patients with idiopathic VTE is controversial. MATERIALS AND METHODS: Systemic review and meta-analysis of all available prospective trials comparing extensive to limited screening for occult malignancies in patients with idiopathic VTE. PRIMARY OUTCOME: all-cause mortality. SECONDARY OUTCOMES: cancer related mortality, early cancer diagnosis, cancer diagnosis at the end of follow up and cancer diagnosis at an early stage. Risk ratios (RR) with 95% confidence intervals (CIs) were estimated and pooled. RESULTS: The study included five trials and 2287 patients. Extensive screening did not affect all-cause mortality at the end of follow up [RR 0.86 (95% CI 0.58-1.27)] or cancer-related mortality [RR 0.93 (95% CI 0.54-1.58)]. Yet, it yielded more diagnoses of cancer [RR 2.17 (95% CI 1.42-3.32)]. Rates of cancer diagnosis at an early stage did not differ statistically between the two groups [RR 1.49 (95% CI 0.86-2.56)]. However, analysis of the randomized controlled trials alone showed a tendency towards early stage cancer at diagnosis in extensive screening group in, with results almost statistically significant [RR 2.14 (95% CI 0.98-4.67), p=0.06]. CONCLUSIONS: Extensive screening for malignancy after idiopathic VTE does not affect mortality rates. Yet, it yields more cancer diagnoses shortly after the VTE event. Further research is needed to determine whether extensive screening might be proper for specific high risk populations.

Yield of workup for patients with idiopathic presentation of the syndrome of inappropriate antidiuretic hormone secretion.

PURPOSE: To determine the proportion of patients for whom the syndrome of inappropriate antidiuretic hormone secretion (SIADH) is the presenting symptom of an underlying disorder, to describe the yield of different diagnostic modalities for patients with SIADH and an unknown etiology, and to define patients for whom such a workup is indicated. METHODS: A single center retrospective study including all patients diagnosed with SIADH without an apparent etiology in a large community hospital and tertiary center between 1.1.07 and 1.1.13. Two physicians reviewed every patient's medical file for predetermined relevant clinical data. RESULTS: Eleven of the 99 patients without an apparent etiology for SIADH at presentation were found to have an underlying cause on workup. Yield of performed workup was low, with a pathology demonstrated on 0%-30.8% of tests according to the different modalities used. Patients with presumed idiopathic SIADH at presentation who were later found to have a specific etiology were younger than patients with true idiopathic SIADH, had a significantly shorter duration of hyponatremia prior to SIADH diagnosis, had higher urine osmolality and a clinical presentation suggestive of an undiagnosed disorder. CONCLUSIONS: Our findings support a clinically-based approach to patients with idiopathic SIADH, rather than an extensive routine workup for all patients.

Acute promyelocytic leukemia with isochromosome 17q and cryptic PML-RARA successfully treated with all-trans retinoic acid and arsenic trioxide.

Acute promyelocytic leukemia (APL) is a subtype of acute leukemia that is characterized by typical morphology, bleeding events and distinct chromosomal aberrations, usually the t(15;17)(q22;q21) translocation. Approximately 9% of APL patients harbor other translocations involving chromosome 17, such as the t(11;17)(q23;q21), t(5;17)(q35;q12-21), t(11;17)(q13;q21), and der(17). All-trans retinoic acid (ATRA) and arsenic trioxide (ATO) have specific targeted activities against the PML-RARA fusion protein. The combination of ATRA and ATO is reportedly superior to chemotherapy and ATRA as induction therapy for APL. The clinical significance of non-t(15:17) APL-related aberrations is controversial, with conflicting reports regarding sensitivity to modern, targeted therapy. Isochromosome 17q (iso(17q)) is rarely associated with APL and usually occurs concurrently with the t(15:17) translocation. No published data is available regarding the efficacy of ATO-based therapy for APL patients who harbor iso(17q). We report on an APL patient with iso(17q) as the sole cytogenetic aberration and a cryptic PML-RARA transcript, who was treated with ATRA and ATO after failure of chemotherapy and achieved complete remission. To our knowledge, this is the first published report of APL associated with iso(17q) as the sole cytogenetic aberration, which was successfully treated with an ATO containing regimen.

The syndrome of inappropriate antidiuretic hormone secretion: Distribution and characterization according to etiologies.

PURPOSE: To determine the distribution of etiologies for the syndrome of inappropriate antidiuretic hormone secretion (SIADH) in hospitalized patients and to characterize patients according to the different etiologies. METHODS: A single-center retrospective study including all patients diagnosed with SIADH in a large community hospital and tertiary center between 1.1.2007 and 1.1.2013. Two physicians reviewed every patient's medical file for predetermined relevant clinical data. RESULTS: The study cohort included 555 patients. The most common etiologies were malignancies and medication-induced SIADH, followed by idiopathic SIADH, pulmonary infections, pain and nausea, and central nervous system (CNS) disorders. Subgroup analysis according to etiology showed that CNS disorders were associated with more severe episodes of SIADH. Patients with idiopathic SIADH were older than patients with a specific diagnosis, had a lower urine osmolality, and required less treatment with hypertonic saline. Long-term survival was determined primarily by SIADH etiology rather than hyponatremia severity, with hazard ratios for death of up to 7.31 (95% CI 4.93-10.82, p<0.001) for patients with malignancy-associated SIADH as compared to patients with idiopathic SIADH. Hyponatremia grade at short-term follow-up was also predictive for long-term survival (HR 1.42 per grade, 95% CI 1.21-1.66, p<0.001). CONCLUSIONS: Patients with SIADH have different characteristics and a different prognosis according to SIADH etiology. Serum sodium concentration at short-term follow-up is predictive of long-term survival. These findings might have diagnostic and treatment-related implications.