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Introduction: Biliopancreatic diversion with duodenal switch (BPD-DS) has often been reserved for patients with BMI > 50 kg/m 2 . We aim to assess the safety of BPD-DS in patients with morbid obesity (BMI 3 35 kg/m 2 and < 50 kg/m 2 ) using a 150-cm common channel (CC), 150-cm Roux limb, and 60-fr bougie., Methods: A retrospective review was performed on patients with a BMI < 50 mg/k 2 who underwent a BPD-DS in 2016-2019 at a single institution. Limb lengths were measured with a laparoscopic instrument with minimal tension. Sleeve gastrectomy was created with 60-fr bougie. Variables were compared using paired t test, Chi-square analysis or repeated measures ANOVA where appropriate., Results: Forty-five patients underwent BPD-DS. CC lengths and Roux limb lengths were 158 ± 20 cm and 154 ± 18 cm, respectively. Preoperative BMI was 44.9 ± 2.3 kg/m 2 and follow-up was 2.7 ± 1.4 years. One patient required reoperation for bleeding and died from multiorgan failure and delayed sleeve leak. There was 1 (2.2%) readmission for contained anastomotic leak and 2 ED visits (4.5%) within 30 days. There were no marginal ulcers, limb length revisions, or need for parental nutrition. Percent excess weight loss was 67.2 ± 19.7%. 88.9% (N = 8), 86.6% (N = 13), and 55.5% (N = 5) of patients had resolution or improvement of their diabetes mellitus type II, hypertension, and hyperlipidemia, respectively. 40% (N = 4) of patients had resolution of their gastroesophageal reflux disease (GERD) and 11.4% (N = 5) developed de novo GERD. 32% (N = 14) of patients had vitamin D deficiency and 25% (N = 11) experienced zinc deficiency., Conclusion: BPD-DS may be considered in patients with BMI < 50 kg/m 2 with 150-cm CC, 150-cm Roux limb, and a 60-fr bougie sleeve gastrectomy. There was sustained weight loss and no protein calorie malnutrition, but Vitamin D and zinc deficiency remained a challenge. Careful patient selection and proper counseling of the risks and benefits are necessary., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Introduction: Post-operative gastroesophageal reflux disease (GERD) remains a significant morbidity following sleeve gastrectomy (SG). We aim to evaluate the incidence and impact within a single center experience., Materials and Methods: A retrospective review of a prospectively maintained database was performed identifying laparoscopic or robotic SG patients. Primary outcomes included weight loss, rates of post-operative GERD (de-novo or aggravated), and re-intervention. Subgroup analysis was performed between patients with (Group 1) and without (Group 2) post-operative GERD. De-novo GERD and aggravated was defined as persistent GERD complaints or new/increased PPI usage in GERD naive or prior GERD patients, respectively., Results: 392 patients were identified between 2014 and 2019. Average demographics: age 42.3 (18-84) years, Charlson Comorbidity Index (CCI) 1.12 (0-10), and body mass index (BMI) 47.7 (28-100). 98% were performed laparoscopically. Average excess weight loss (EWL) was 51.0% and 46.4% at 1 and 2 years post-operatively. Average follow up was 516 (6-2694) days. 69 (17%) patients developed post operative de-novo or aggravated GERD. Group 1 had significantly higher EWL at 9 months (57% vs 47%, p 0.003). 13 (3%) patients required operative re-intervention for GERD and other morbidities: 4 RYGB conversions, 4 diagnostic laparoscopies, 3 HHR, 1 MSA placement. Group 1 had higher rates of post-operative intervention (14% vs 1%, p 0.0001). Subanalysis demonstrated that Group 1 had elevated preoperative DeMeester scores on pH testing (34.8 vs 18.9, p 0.03). De-novo GERD had an elevated post-operative total acid exposure when compared to aggravated GERD (12.7% vs 7.0% p 0.03). No significant differences were found between preoperative endoscopy findings, pre and postoperative total acid exposure, post-operative DeMeester scores, and high-resolution manometry values regarding de-novo/aggravated GERD development., Conclusion: Preoperative DeMeester scores may serve as risk indicators regarding post-operative GERD. Outcomes such as reintervention remain elevated in post-operative GERD patients., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: The legalization of cannabis in several states has led to increased documented use in the population. Bariatric surgery patients are no exception with estimates of anywhere from 6 to 8%. Cannabis is known to be associated with increased appetite, mood disorders, hyperphagia, and rarely, hyperemesis, which can potentially affect post-surgical weight loss. We aim to study the differences in bariatric surgery outcomes between cannabis users and non-users., Methods: A retrospective review identified patients undergoing bariatric surgery. Patients were divided into two groups, cannabis users (CU) and non-cannabis users (non-CU). Cannabis users (defined as using at least once weekly) and a group of non-users were called to obtain additional information. Primary outcome was weight loss. Secondary outcomes included incidence of post-operative nausea and vomiting (PONV), length of stay (LOS), readmission, and need for additional intervention., Results: A cohort of 364 sleeve gastrectomy patients met inclusion criteria, 31 (8.5%) CU and 333 (91.5%) non-CU. There was no difference in EWL between CU and non-CU at 1 week, 1 month, 3 months, 6 months, 9 months, 1 year, and 2 years. However, the CU group trended towards greater EWL at 3 years (52.9% vs. 38.1%, p = 0.094) and at 5 years (49.8% vs. 32.7%, p = 0.068). There were no significant differences between CU and non-CU with respect to either incidence or severity of PONV at one year after surgery or longer follow-up., Conclusion: Cannabis users did not experience inferior weight loss after bariatric surgery despite common assumptions that appetite stimulation can lead to suboptimal weight loss outcomes. Our findings add to other work challenging this dogma. Larger, long-term, multicenter studies are warranted., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Background: The majority of laparoscopic paraesophageal hernia (PEH) repairs are performed electively. We aimed to investigate the frequency of non-elective laparoscopic (MIS) PEH repair and compare 30-day outcomes to elective MIS repairs and non-elective open repairs. We hypothesized that an increasing percentage of non-elective PEH repairs would be performed laparoscopically and that this population would have improved outcomes compared to non-elective open PEH counterparts., Methods: The 2011-2016 NSQIP PUFs were used to identify patients who underwent PEH repair. Case status was classified as open vs. MIS and elective versus non-elective. Preoperative patient characteristics, operative details, discharge destination, and 30-day postoperative complication rates were compared. Logistic regression was used to examine the impact of case status on 30-day mortality., Results: We identified 20,010 patients who underwent PEH. There were an increasing number of MIS PEH repairs in NSQIP between 2011 and 2016. Non-elective repairs were performed in 2,173 patients and 73.4% of these were completed laparoscopically. Elective MIS patients were younger, had a higher BMI, and were more likely to be functionally independent (p < 0.01) than their non-elective counterparts. Non-elective MIS patients had a higher wound class and ASA class compared to their elective counterparts. Compared to elective MIS cases, non-elective MIS PEH repair was associated with increased odds of mortality, even after controlling for patient characteristics (OR = 1.76, p = 0.02). There was no statistically significant difference in mortality for non-elective MIS vs. non-elective open PEH repair. There is an increase in non-elective PEH repairs recorded in NSQIP over time studied., Conclusions: The population undergoing non-elective MIS PEH repairs is different from their elective MIS counterparts and experience a higher postoperative mortality rate. While the observed increased utilization of MIS techniques in non-elective PEH repairs likely provides benefits for the patient, there remain differences in outcomes for these patients compared to elective PEH repairs.

The aerosol release during the professional application of two different isocyanate based two component spray systems was identified and the physicochemical properties of the released airborne aerosols were characterized. For this purpose, aerosol release fractions were measured using a mass balance method described by Schwarz and Koch. Besides the release of total aerosol mass special emphasis was directed to the content of free monomeric MDI (4,4'- and 2,4'-diphenylmethane diisocyanate) in three particle size fractions relevant for inhalation uptake: inhalable, thoracic, and respirable size fraction. Two products were investigated: a two component PUR (polyurethane) spray foam (Elastopor) and a polyurea spray coating (Elastocoat). The mass fraction of the applied products released with the overspray as inhalable aerosol is 6.3 × 10-4 (Elastopor) and 4.0 × 10-4 (Elastocoat). Of the released total overspray aerosol 75 or 80% were in the thoracic size range, and 26 or 47% in the respirable regime for the PUR spray foam or the polyurea spray coating, respectively. At the time point of release the content of monomeric MDI in the aerosol corresponds to the composition of the bulk product. However, analysis of air samples indicates that <1% of the spray foam aerosol mass release fraction is attributed to free monomeric 4,4'- and 2,4'-MDI. For the Spray Coating the monomeric MDI fraction is <0.1%. Higher oligomers of MDI and prereacted oligomeric reaction products make up a few percent of the aerosol. This results in a total fraction of 0.0023% (spray foam) and 0.00015% (spray coating), respectively, of the sprayed monomeric MDI that is transferred into an inhalable aged aerosol. This data demonstrates, that during professional spraying only a small fraction of the total applied mass is released as airborne aerosol. The potential distribution of the theoretically inhalable aerosol in the respiratory tract and a low residual monomer content is described, significantly contributing to a refined safety assessment of the spray applications at the workplaces., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Occupational Hygiene Society.)

A variety of literature addresses the question of how the age distribution of deaths changes over time as life expectancy increases. However, corresponding terms such as extension, compression, or rectangularization are sometimes defined only vaguely, and statistics used to detect certain scenarios can be misleading. The matter is further complicated because mixed scenarios can prevail, and the considered age range can have an impact on observed mortality patterns. In this article, we establish a unique classification framework for realized mortality scenarios that allows for the detection of both pure and mixed scenarios. Our framework determines whether changes of the deaths curve over time show elements of extension or contraction; compression or decompression; left- or right-shifting mortality; and concentration or diffusion. The framework not only can test the presence of a particular scenario but also can assign a unique scenario to any observed mortality evolution. Furthermore, it can detect different mortality scenarios for different age ranges in the same population. We also present a methodology for the implementation of our classification framework and apply it to mortality data for U.S. females.

The NewProt protein engineering portal is a one-stop-shop for in silico protein engineering. It gives access to a large number of servers that compute a wide variety of protein structure characteristics supporting work on the modification of proteins through the introduction of (multiple) point mutations. The results can be inspected through multiple visualizers. The HOPE software is included to indicate mutations with possible undesired side effects. The Hotspot Wizard software is embedded for the design of mutations that modify a proteins' activity, specificity, or stability. The NewProt portal is freely accessible at http://newprot.cmbi.umcn.nl/ and http://newprot.fluidops.net/., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Pyridoxal-5'-phosphate (PLP)-dependent enzymes are ubiquitous in nature and catalyze a variety of important metabolic reactions. The fold-type III PLP-dependent enzyme family is primarily comprised of decarboxylases and alanine racemases. In the development of a multiple structural alignment database (3DM) for the enzyme family, a large subset of 5666 uncharacterized proteins with high structural, but low sequence similarity to alanine racemase and decarboxylases was found. Compared to these two classes of enzymes, the protein sequences being the object of this study completely lack the C-terminal domain, which has been reported important for the formation of the dimer interface in other fold-type III enzymes. The 5666 sequences cluster around four protein templates, which also share little sequence identity to each other. In this work, these four template proteins were solubly expressed in Escherichia coli, purified, and their substrate profiles were evaluated by HPLC analysis for racemase activity using a broader range of amino acids. They were found active only against alanine or serine, where they exhibited Michaelis constants within the range of typical bacterial alanine racemases, but with significantly lower turnover numbers. As the already described racemases were proposed to be active and appeared to be monomers as judged from their crystal structures, we also investigated this aspect for the four new enzymes. Here, size exclusion chromatography indicated the presence of oligomeric states of the enzymes and a native-PAGE in-gel assay showed that the racemase activity was present only in an oligomeric state but not as monomer. This suggests the likelihood of a different behavior of these enzymes in solution compared to the one observed in crystalline form.

Baeyer-Villiger monooxygenases (BVMOs) catalyze the oxidation of ketones to esters or lactones by using molecular oxygen and a cofactor. Type I BVMOs display a strong preference for NADPH. However, for industrial purposes NADH is the preferred cofactor, as it is ten times cheaper and more stable. Thus, we created a variant of the cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 (CHMO Acineto ); this used NADH 4200-fold better than NADPH. By combining structure analysis, sequence alignment, and literature data, 21 residues in proximity of the cofactor were identified and targeted for mutagenesis. Two combinatorial variants bearing three or four mutations showed higher conversions of cyclohexanone with NADH (79 %) compared to NADPH (58 %) as well as specificity. The structural reasons for this switch in cofactor specificity of a type I BVMO are especially a hydrogen-bond network coordinating the two hydroxy groups of NADH through direct interactions and bridging water molecules., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The use of transaminases to access pharmaceutically relevant chiral amines is an attractive alternative to transition-metal-catalysed asymmetric chemical synthesis. However, one major challenge is their limited substrate scope. Here we report the creation of highly active and stereoselective transaminases starting from fold class I. The transaminases were developed by extensive protein engineering followed by optimization of the identified motif. The resulting enzymes exhibited up to 8,900-fold higher activity than the starting scaffold and are highly stereoselective (up to >99.9% enantiomeric excess) in the asymmetric synthesis of a set of chiral amines bearing bulky substituents. These enzymes should therefore be suitable for use in the synthesis of a wide array of potential intermediates for pharmaceuticals. We also show that the motif can be engineered into other protein scaffolds with sequence identities as low as 70%, and as such should have a broad impact in the field of biocatalytic synthesis and enzyme engineering.

The regioselectivity of the Baeyer-Villiger monooxygenase-catalyzed oxidation is governed mostly by electronic effects leading to the migration of the higher substituted residue. However, in some cases, substrate binding occurs in a way that the less substituted residue lies in an antiperiplanar orientation to the peroxy bond in the Criegee intermediate yielding in the formation of the "abnormal" lactone product. We are the first to demonstrate a complete switch in the regioselectivity of the BVMO from Arthrobacter sp. (CHMOArthro) as exemplified for (+)-trans-dihydrocarvone by redesigning the active site of the enzyme. In the designed triple mutant, the substrate binds in an inverted orientation leading to a ratio of 99:1 in favor of the normal lactone instead of exclusive formation of the abnormal lactone in case of the wild type enzyme. In order to validate our computational study, the beneficial mutations were successfully transferred to the CHMO from Acinetobacter sp. (CHMOAcineto), again yielding in a complete switch of regioselectivity.

Baeyer-Villiger monooxygenases (BVMO) belong to the class B of flavin-dependent monooxygenases (type I BVMOs) and catalyze the oxidation of (cyclic) ketones into esters and lactones. The prototype BVMO is the cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871. This enzyme shows an impressive substrate scope with a high chemo-, regio- and/or enantioselectivity. BVMO reactions are often difficult, if not impossible to achieve by chemical approaches and this makes these enzymes thus highly desired candidates for industrial applications. Unfortunately, the industrial use is hampered by several factors related to the lack of stability of these biocatalysts. Thus, the aim of this study was to improve the CHMO's long-term stability, one of the most relevant parameter for biocatalytic processes, and additionally its stability against oxidation. We used an easy computational method for the prediction of stabilizing disulfide bonds in the CHMO-scaffold. The three most promising predicted disulfide pairs were created and biochemically characterized. The most oxidatively stable variant (Y411C-A463C) retained nearly 60% activity after incubation with 25 mM H2O2 whereas the wild type retained only 16%. In addition, one extra disulfide pair (T415C-A463C) was created and tested for increased stability. The melting temperature (Tm) of this variant was increased by 5°C with simultaneous improved long-term stability. After verification by ABD-F labeling that this mutant does not form a disulfide bond, single and double Cys/Ser mutants were prepared and investigated. Subsequent analysis revealed that the T415C single point variant is the most stable variant with a 30-fold increased long-term stability (33% residual activity after 24h incubation at 25°C) showcasing a great achievement for practical applications., (Copyright © 2015 Elsevier B.V. All rights reserved.)

To alter the amine donor/acceptor spectrum of an (S)-selective amine transaminase (ATA), a library based on the Vibrio fluvialis ATA targeting four residues close to the active site (L56, W57, R415 and L417) was created. A 3DM-derived alignment comprising fold class I pyridoxal-5'-phosphate (PLP)-dependent enzymes allowed identification of positions, which were assumed to determine substrate specificity. These positions were targeted for mutagenesis with a focused alphabet of hydrophobic amino acids to convert an amine:α-keto acid transferase into an amine:aldehyde transferase. Screening of 1200 variants revealed three hits, which showed a shifted amine donor/acceptor spectrum towards aliphatic aldehydes (mainly pentanal), as well as an altered pH profile. Interestingly, all three hits, although found independently, contained the same mutation R415L and additional W57F and L417V substitutions.

The unique ribonuclease S (RNase S) system, derived from proteolytic cleavage of bovine ribonuclease A (RNase A), consists of a tight complex formed by a peptide (amino acids 1-20) and a protein (21-124) part. These fragments, designated as S-peptide and S-protein, can be separated by two purification steps. By addition of synthetic S-peptide derivatives to the S-protein, semisynthetic RNase S is reassembled with high efficiency. Based on this peptide-protein complementation noncanonical amino acids can be easily introduced into a protein host. Here we describe the preparation of the S-protein from RNase A as well as the characterization of the reassembled semisynthetic RNase S complex. Complex formation can be monitored by RNase activity, circular dichroism, or fluorescence polarization. Structure-based enzyme design of the RNase S scaffold is possible based on high-resolution crystal structures of RNase S and its semisynthetic variants.

Huntington's disease (HD) is a neurodegenerative disorder that affects approximately 1 in 10 000 individuals. The underlying gene mutation was identified as a CAG-triplet repeat expansion in the gene huntingtin. The CAG sequence codes for glutamine, and in HD, an expansion of the polyglutamine (poly-Q) stretch above 35 glutamine residues results in pathogenicity. It has been demonstrated in various animal models that only the expression of exon 1 huntingtin, a 67-amino acid-long polypeptide plus a variable poly-Q stretch, is sufficient to cause full HD-like pathology. Therefore, a deeper understanding of exon 1 huntingtin, its structure, aggregation mechanism and interaction with other proteins is crucial for a better understanding of the disease. Here, we describe the synthesis of a 109-amino acid-long exon 1 huntingtin peptide including a poly-Q stretch of 42 glutamines. This microwave-assisted solid phase peptide synthesis resulted in milligram amounts of peptide with high purity. We also synthesized a nonpathogenic version of exon 1 huntingtin (90-amino acid long including a poly-Q stretch of 23 glutamine residues) using the same strategy. In circular dichroism spectroscopy, both polypeptides showed weak alpha-helical properties with the longer peptide showing a higher helical degree. These model peptides have great potential for further biomedical analyses, e.g. for large-scale pre-screenings for aggregation inhibitors, further structural analyses as well as protein-protein interaction studies.

Objective: To evaluate 24-week virologic effectiveness of novel antiretroviral regimens for treatment of three-class experienced adult patients in a clinical practice setting following the US Food and Drug Administration approval of darunavir (DRV) for this population., Design: A prospective cohort study., Setting: A single-center, academic HIV clinic., Participants: Three-class antiretroviral-experienced patients changing regimens between July 2006 and May 2008. Sociodemographic, psychosocial, and clinical characteristics were collected at baseline and during prospective follow-up., Outcome Measures: Plasma HIV viral load below 50 copies/ml and change in CD4 cell count at 24 weeks following regimen change. The Stanford Genotype Database was used to analyze HIV genotype resistance results and determine the number of active drugs in each regimen. Multivariate models and propensity score methods were employed to assess outcome measures., Results: Among 109 three-class experienced patients, who previously received an average of 10.5 prior antiretrovirals, 55% achieved viral load below 50 copies/ml at 24 weeks. Treatment strategy was classified as nonprotease inhibitor (n = 25), DRV/ritonavir (DRV/r) (n = 51), or other protease inhibitor (n = 33). The number of active drugs was not significantly different across strategies (P = 0.24). In multivariate analysis, patients treated with DRV/r (65%, odds ratio = 4.24 vs. nonprotease inhibitor strategy, 95% confidence interval = 1.28-14.06), raltegravir (65%, odds ratio = 3.10, 95% confidence interval = 1.12-8.62), or both were more likely to achieve viral load below 50 copies/ml., Conclusion: Among antiretroviral-experienced patients changing regimens, those treated with DRV/r, raltegravir, or both were more likely to achieve a viral load below 50 copies/ml at 24 weeks. The effectiveness of these agents in routine clinical care mirrors their efficacy in clinical trials and has ushered in a new era in the therapy of three-class experienced patients.

The fourth survey by standardized questionnaire in November 1994 (response rate: 71.6%, n = 308, mean age: 68.1 years) to identify effects of the changes in the eastern German cultural and social scene among senior students enrolled at the Martin Luther University Halle-Wittenberg since 1989 (HALSEKO) revealed a distinct deterioration in intergenerational relations which has been interpreted in terms of Mead's theoretical conception of the induction of intergenerational conflict by the abrupt change in cultural values in the new Federal states. Negative assessment were given by 69.5% (1992: 33.4%). Statistical analysis by bivariant contingency tables yielded an assessment pattern with two dimensions. These consisted personal discrimination by younger people and changes in intrafamilial intergenerational relations owing to fewer contacts, stress due to the job situation for children and grandchildren, tension within the family and anxiety at the thought of loneliness. The latter causes have a hurtfull effect. The list of possible causes for the deterioration in intergenerational relations that formed part of the questionnaire showed that most subjects considered unemployment (71.3%) and changes in interpersonal contact (71.1%) to be responsible, the latter being described as a situation in which people do not have time for each other. In view of the importance of social relations for the well-being of the elderly, relations between the generations should remain on the agenda as a subject for further research and preventive conceptions.