The pathway for coenzyme M biosynthesis in bacteria.

Mercaptoethane sulfonate or coenzyme M (CoM) is the smallest known organic cofactor and is most commonly associated with the methane-forming step in all methanogenic archaea but is also associated with the anaerobic oxidation of methane to CO2 in anaerobic methanotrophic archaea and the oxidation of short-chain alkanes in Syntrophoarchaeum species. It has also been found in a small number of bacteria capable of the metabolism of small organics. Although many of the steps for CoM biosynthesis in methanogenic archaea have been elucidated, a complete pathway for the biosynthesis of CoM in archaea or bacteria has not been reported. Here, we present the complete CoM biosynthesis pathway in bacteria, revealing distinct chemical steps relative to CoM biosynthesis in methanogenic archaea. The existence of different pathways represents a profound instance of convergent evolution. The five-step pathway involves the addition of sulfite, the elimination of phosphate, decarboxylation, thiolation, and the reduction to affect the sequential conversion of phosphoenolpyruvate to CoM. The salient features of the pathway demonstrate reactivities for members of large aspartase/fumarase and pyridoxal 5'-phosphate-dependent enzyme families.

Transcarotid Arterial Revascularization of Symptomatic Internal Carotid Artery Disease: A Systematic Review and Study-Level Meta-Analysis.

BACKGROUND: Transcarotid artery revascularization (TCAR) is an interventional therapy for symptomatic internal carotid artery disease. Currently, the utilization of TCAR is contentious due to limited evidence. In this study, we evaluate the safety and efficacy of TCAR in patients with symptomatic internal carotid artery disease compared with carotid endarterectomy (CEA) and carotid artery stenting (CAS). METHODS: A systematic review was conducted, spanning from January 2000 to February 2023, encompassing studies that used TCAR for the treatment of symptomatic internal carotid artery disease. The primary outcomes included a 30-day stroke or transient ischemic attack, myocardial infarction, and mortality. Secondary outcomes comprised cranial nerve injury and major bleeding. Pooled odds ratios (ORs) for each outcome were calculated to compare TCAR with CEA and CAS. Furthermore, subgroup analyses were performed based on age and degree of stenosis. In addition, a sensitivity analysis was conducted by excluding the vascular quality initiative registry population. RESULTS: A total of 7 studies involving 24 246 patients were analyzed. Within this patient cohort, 4771 individuals underwent TCAR, 12 350 underwent CEA, and 7125 patients underwent CAS. Compared with CAS, TCAR was associated with a similar rate of stroke or transient ischemic attack (OR, 0.77 [95% CI, 0.33-1.82]) and myocardial infarction (OR, 1.29 [95% CI, 0.83-2.01]) but lower mortality (OR, 0.42 [95% CI, 0.22-0.81]). Compared with CEA, TCAR was associated with a higher rate of stroke or transient ischemic attack (OR, 1.26 [95% CI, 1.03-1.54]) but similar rates of myocardial infarction (OR, 0.9 [95% CI, 0.64-1.38]) and mortality (OR, 1.35 [95% CI, 0.87-2.10]). CONCLUSIONS: Although CEA has traditionally been considered superior to stenting for symptomatic carotid stenosis, TCAR may have some advantages over CAS. Prospective randomized trials comparing the 3 modalities are needed.

Unravelling the complex speciation of halozincate ionic liquids using X-ray spectroscopies and calculations.

Using a combination of liquid-phase X-ray spectroscopy experiments and small-scale calculations we have gained new insights into the speciation of halozincate anions in ionic liquids (ILs). Both core and valence X-ray photoelectron spectroscopy (XPS) experiments were performed directly on the liquid-phase ILs, supplemented by Zn 1s X-ray absorption near edge structure (XANES) spectroscopy. Density functional theory (DFT) calculations were carried out on both 1- and 2- halozincate anions, in both a generalised solvation model SMD (Solvation Model based on Density) and the gas phase, to give XP spectra and total energy differences; time-dependent DFT was used to calculate XANES spectra. Speciation judgements were made using a combination of the shape and width of the experimental spectra, and visual matches to the calculated spectra. For 2- halozincate anions, excellent matches were found between the experimental and calculated XP spectra, clearly showing that only 2- halozincate anions were present at all zinc halide mole fractions, x, studied. At specific values of x (0.33, 0.50, 0.60) only one halozincate anion was present; equilibria of different halozincate anions at those values of x were not observed. All findings show that chlorozincate anion and bromozincate anion speciation matched at the same x. Based on the results, predictions are made of the halozincate anion speciation for all values of x up to 0.67. Caution is advised when using differences in calculated total energies obtained from DFT to judge halozincate anion speciation, even when the SMD was employed, as predictions based on total energy differences did not always match the findings from the experimental and calculated spectra. Our findings clearly establish that the combination of high-quality experimental data from multiple spectroscopies and a wide range of calculated structures are essential to have high confidence in halozincate anion speciation identification.

Pichia kudriavzevii as feed additive in Nile tilapia (Oreochromis niloticus) diet.

Yeasts are unicellular eukaryotic microorganisms extensively employed in various applications, notably as an alternative source of protein in feeds, owing to their nutritional benefits. Despite their potential, marine and mangrove yeast species used in the aquaculture industry have received little attention in the Philippines. Pichia kudriavzevii (A2B R1 ISO 3), sourced from bark samples, was selected and mass-produced due to its high protein content and amino acid profile. The dried biomass of P. kudriavzevii was incorporated into the diets of Nile tilapia (Oreochromis niloticus) juveniles at varying inclusion levels (0, 1, 2, and 4 g/kg diet) and its effect on their growth performance, body composition, and liver and intestinal morphology was assessed after 40 days of feeding. The groups that received P. kudriavzevii at a concentration of 2 g/kg diet exhibited higher final body weight, percent weight gain, and specific growth rate in comparison to the other treatment groups. Whole body proximate composition did not vary among the dietary groups. Intestinal and liver histopathology also indicated no abnormalities. These findings suggest the potential of ascomycetous P. kudriavzevii as a beneficial feed additive in Nile tilapia diets, warranting further investigation into its long-term effects and broader applications in fish culture.

Imaging in gynecological disease (26): clinical and ultrasound characteristics of benign retroperitoneal pelvic peripheral-nerve-sheath tumors.

OBJECTIVE: To describe the clinical and sonographic characteristics of benign, retroperitoneal, pelvic peripheral-nerve-sheath tumors (PNSTs). METHODS: This was a retrospective study of patients with a benign, retroperitoneal, pelvic PNST who had undergone preoperative ultrasound examination at a single gynecologic oncology center between 1 January 2018 and 31 August 2022. All ultrasound images, videoclips and final histological specimens of benign PNSTs were reviewed side-by-side in order to: describe the ultrasound appearance of the tumors, using the terminology of the International Ovarian Tumor Analysis (IOTA), Morphological Uterus Sonographic Assessment (MUSA) and Vulvar International Tumor Analysis (VITA) groups, following a predefined ultrasound assessment form; describe their origin in relation to nerves and pelvic anatomy; and assess the association between their ultrasound features and histotopography. A review of the literature reporting benign, retroperitoneal, pelvic PNSTs with preoperative ultrasound examination was performed. RESULTS: Five women (mean age, 53 years) with a benign, retroperitoneal, pelvic PNST were identified, four with a schwannoma and one with a neurofibroma, of which all were sporadic and solitary. All patients had good-quality ultrasound images and videoclips and final biopsy of surgically excised tumors, except one patient managed conservatively who had only a core needle biopsy. In all cases, the findings were incidental. The five PNSTs ranged in maximum diameter from 31 to 50 mm. All five PNSTs were solid, moderately vascular tumors, with non-uniform echogenicity, well-circumscribed by hyperechogenic epineurium and with no acoustic shadowing. Most of the masses were round (n = 4 (80%)), and contained small, irregular, anechoic, cystic areas (n = 3 (60%)) and hyperechogenic foci (n = 5 (100%)). In the woman with a schwannoma in whom surgery was not performed, follow-up over a 3-year period showed minimal growth (1.5 mm/year) of the mass. We also summarize the findings of 47 cases of benign retroperitoneal schwannoma and neurofibroma identified in a literature search. CONCLUSIONS: On ultrasound examination, no imaging characteristics differentiate reliably between benign schwannomas and neurofibromas. Moreover, benign PNSTs show some similar features to malignant retroperitoneal tumors. They are solid lesions with intralesional blood vessels and show degenerative changes such as cystic areas and hyperechogenic foci. Therefore, ultrasound-guided biopsy may play a pivotal role in their diagnosis. If confirmed to be benign PNSTs, these tumors can be managed conservatively, with ultrasound surveillance. © 2023 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Complications associated with subaxial placement of pedicle screws versus lateral mass screws in the cervical spine (C2-T1): systematic review and meta-analysis comprising 4,165 patients and 16,669 screws.

Lateral mass screw (LMS) and cervical pedicle screw (CPS) fixation are among the most popular techniques for posterior fusion of the cervical spine. Early research prioritized the LMS approach as the trajectory resulted in fewer neurovascular complications; however, with the incorporation of navigation assistance, the CPS approach should be re-evaluated. Our objective was to report the findings of a meta-analysis focused on comparing the LMS and CPS techniques in terms of rate of various complications with inclusion of all levels from C2 to T1. We conducted a systematic review of PubMed and EMBASE databases with final inclusion criteria focused on identifying studies that reported outcomes and complications for either the CPS or LMS technique. These studies were then pooled, and statistical analyses were performed from the cumulative data. A total of 60 studies comprising 4165 participants and 16,669 screws placed within the C2-T1 levels were identified. Within these studies, the LMS group had a significantly increased odds for lateral mass fractures (odds ratio [OR] = 43.2, 95% confidence interval [CI] = 2.62-711.42), additional cervical surgeries (OR = 5.56, 95%CI = 2.95-10.48), and surgical site infections (SSI) (OR = 5.47, 95%CI = 1.65-18.16). No other significant differences between groups in terms of complications were identified. Within the subgroup analysis of navigation versus non-navigation-guided CPS placement, no significant differences were identified for individual complications, although collectively significantly fewer complications occurred with navigation (OR = 5.29, 95%CI = 2.03-13.78). The CPS group had significantly fewer lateral mass fractures, cervical revision surgeries, and SSIs. Furthermore, navigation-assisted CPS placement was associated with a significant reduction in complications overall.

Lateral ear canal resection and bulla osteotomy with marsupialization to treat otitis media in rabbits: forty-eight procedures.

OBJECTIVE: Describe lateral ear canal resection and bulla osteotomy with marsupialization (LECARBOM) in rabbits with otitis media (OM), and report outcomes, complications, bacteria cultured from middle ears, and their antimicrobial susceptibility testing (AST) results. STUDY DESIGN: Retrospective clinical case series; single referral hospital. ANIMALS: Forty-two rabbits with naturally occurring OM. METHODS: Medical records (2011-2021) of rabbits with CT-confirmed OM undergoing surgery were reviewed for outcomes, complications, bacteria cultured from middle ears, and AST. RESULTS: Surgery was performed on 48 ears, and outcomes determined 21 days postoperatively. All rabbits survived the procedure. Otitis externa resolved in all lop-eared rabbits. Of 29 ears with OM-associated head tilt, eight (28%) resolved, nine (31%) improved, seven (24%) remained unchanged and five (17%) worsened. Outcome was not associated with head tilt duration nor OM severity. Postoperative complications occurred in 12 (25%) cases, seven of which had wound margin dehiscence that healed by 21 days. The most frequent bacteria isolated were Pasteurella multocida (16%), Bordetella bronchiseptica (14%) and Staphylococcus aureus (14%). Bacteria were sensitive to azithromycin, marbofloxacin or enrofloxacin and resistant to penicillin. CONCLUSION: LECARBOM is a well-tolerated surgical procedure to treat OM. It also improved or stabilized 83% of cases with head tilt and resolved all cases with otitis externa. CLINICAL SIGNIFICANCE: LECARBOM with administration of an AST-sensitive antibiotic, or azithromycin when no AST is performed, should be effective in rabbits with OM. Postsurgically, it resolves or alleviates most secondary conditions such as otitis externa, head tilt and facial nerve paralysis.

The unique Phe-His dyad of 2-ketopropyl coenzyme M oxidoreductase/carboxylase selectively promotes carboxylation and S-C bond cleavage.

The 2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) enzyme is the only member of the disulfide oxidoreductase (DSOR) family of enzymes, which are important for reductively cleaving S-S bonds, to have carboxylation activity. 2-KPCC catalyzes the conversion of 2-ketopropyl-coenzyme M to acetoacetate, which is used as a carbon source, in a controlled reaction to exclude protons. A conserved His-Glu motif present in DSORs is key in the protonation step; however, in 2-KPCC, the dyad is substituted by Phe-His. Here, we propose that this difference is important for coupling carboxylation with C-S bond cleavage. We substituted the Phe-His dyad in 2-KPCC to be more DSOR like, replacing the phenylalanine with histidine (F501H) and the histidine with glutamate (H506E), and solved crystal structures of F501H and the double variant F501H_H506E. We found that F501 protects the enolacetone intermediate from protons and that the F501H variant strongly promotes protonation. We also provided evidence for the involvement of the H506 residue in stabilizing the developing charge during the formation of acetoacetate, which acts as a product inhibitor in the WT but not the H506E variant enzymes. Finally, we determined that the F501H substitution promotes a DSOR-like charge transfer interaction with flavin adenine dinucleotide, eliminating the need for cysteine as an internal base. Taken together, these results indicate that the 2-KPCC dyad is responsible for selectively promoting carboxylation and inhibiting protonation in the formation of acetoacetate.

Recent advances toward the bioconversion of methane and methanol in synthetic methylotrophs.

Abundant natural gas reserves, along with increased biogas production, have prompted recent interest in harnessing methane as an industrial feedstock for the production of liquid fuels and chemicals. Methane can either be used directly for fermentation or first oxidized to methanol via biological or chemical means. Methanol is advantageous due to its liquid state under normal conditions. Methylotrophy, defined as the ability of microorganisms to utilize reduced one-carbon compounds like methane and methanol as sole carbon and energy sources for growth, is widespread in bacterial communities. However, native methylotrophs lack the extensive and well-characterized synthetic biology toolbox of platform microorganisms like Escherichia coli, which results in slow and inefficient design-build-test cycles. If a heterologous production pathway can be engineered, the slow growth and uptake rates of native methylotrophs generally limit their industrial potential. Therefore, much focus has been placed on engineering synthetic methylotrophs, or non-methylotrophic platform microorganisms, like E. coli, that have been engineered with synthetic methanol utilization pathways. These platform hosts allow for rapid design-build-test cycles and are well-suited for industrial application at the current time. In this review, recent progress made toward synthetic methylotrophy (including methanotrophy) is discussed. Specifically, the importance of amino acid metabolism and alternative one-carbon assimilation pathways are detailed. A recent study that has achieved methane bioconversion to liquid chemicals in a synthetic E. coli methanotroph is also briefly discussed. We also discuss strategies for the way forward in order to realize the industrial potential of synthetic methanotrophs and methylotrophs.

Resonant X-ray photoelectron spectroscopy: identification of atomic contributions to valence states.

Valence electronic structure is crucial for understanding and predicting reactivity. Valence non-resonant X-ray photoelectron spectroscopy (NRXPS) provides a direct method for probing the overall valence electronic structure. However, it is often difficult to separate the varying contributions to NRXPS; for example, contributions of solutes in solvents or functional groups in complex molecules. In this work we show that valence resonant X-ray photoelectron spectroscopy (RXPS) is a vital tool for obtaining atomic contributions to valence states. We combine RXPS with NRXPS and density functional theory calculations to demonstrate the validity of using RXPS to identify atomic contributions for a range of solutes (both neutral and ionic) and solvents (both molecular solvents and ionic liquids). Furthermore, the one-electron picture of RXPS holds for all of the closed shell molecules/ions studied, although the situation for an open-shell metal complex is more complicated. The factors needed to obtain a strong RXPS signal are investigated in order to predict the types of systems RXPS will work best for; a balance of element electronegativity and bonding type is found to be important. Additionally, the dependence of RXPS spectra on both varying solvation environment and varying local-covalent bonding is probed. We find that RXPS is a promising fingerprint method for identifying species in solution, due to the spectral shape having a strong dependence on local-covalency but a weak dependence on the solvation environment.

Complications associated with subaxial placement of pedicle screws versus lateral mass screws in the cervical spine: systematic review and meta-analysis comprising 1768 patients and 8636 screws.

Lateral mass screw (LMS) fixation for the treatment of subaxial cervical spine instability or deformity has been traditionally associated with few neurovascular complications. However, cervical pedicle screw (CPS) fixation has recently increased in popularity, especially with navigation assistance, because of the higher pullout strength of the pedicle screws. To their knowledge, the authors conducted the first meta-analysis comparing the complication rates during and/or after CPS and LMS placement for different pathologies causing cervical spine instability. A systematic literature search of PubMed and Embase from inception to January 12, 2021 was performed to identify studies reporting CPS and/or LMS-related complications. Complications were categorized into intraoperative and early postoperative (within 30 days of surgery) and late postoperative (after 30 days from surgery) complications. All studies that met the prespecified inclusion criteria were pooled and cumulatively analyzed. A total of 24 studies were conducted during the time frame of the search and comprising 1768 participants and 8636 subaxially placed screws met the inclusion criteria. The CPS group experienced significantly more postoperative C5 palsy (odds ratio [OR] = 3.48, 95% confidence interval [CI] = 1.27-9.53, p < 0.05). Otherwise, there were no significant differences between the LMS and CPS groups. There were no significant differences between the CPS and LMS groups in terms of neurovascular procedure-related complications other than significantly more C5 palsy in the CPS group.

Adaptive laboratory evolution of methylotrophic Escherichia coli enables synthesis of all amino acids from methanol-derived carbon.

Recent attempts to create synthetic Escherichia coli methylotrophs identified that de novo biosynthesis of amino acids, in the presence of methanol, presents significant challenges in achieving autonomous methylotrophic growth. Previously engineered methanol-dependent strains required co-utilization of stoichiometric amounts of co-substrates and methanol. As such, these strains could not be evolved to grow on methanol alone. In this work, we have explored an alternative approach to enable biosynthesis of all amino acids from methanol-derived carbon in minimal media without stoichiometric coupling. First, we identified that biosynthesis of threonine was limiting the growth of our methylotrophic E. coli. To address this, we performed adaptive laboratory evolution to generate a strain that grew efficiently in minimal medium with methanol and threonine. Methanol assimilation and growth of the evolved strain were analyzed, and, interestingly, we found that the evolved strain synthesized all amino acids, including threonine, from methanol-derived carbon. The evolved strain was then further engineered through overexpression of an optimized threonine biosynthetic pathway. We show that the resulting methylotrophic E. coli strain has a methanol-dependent growth phenotype with homoserine as co-substrate. In contrast to previous methanol-dependent strains, co-utilization of homoserine is not stoichiometrically linked to methanol assimilation. As such, future engineering of this strain and successive adaptive evolution could enable autonomous growth on methanol as the sole carbon source. KEY POINTS: • Adaptive evolution of E. coli enables biosynthesis of all amino acids from methanol. • Overexpression of threonine biosynthesis pathway improves methanol assimilation. • Methanol-dependent growth is seen in minimal media with homoserine as co-substrate.

Understanding and addressing vaccine hesitancy in the context of COVID-19: development of a digital intervention.

OBJECTIVES: Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2) was identified in late 2019, spreading to over 200 countries and resulting in almost two million deaths worldwide. The emergence of safe and effective vaccines provides a route out of the pandemic, with vaccination uptake of 75-90% needed to achieve population protection. Vaccine hesitancy is problematic for vaccine rollout; global reports suggest only 73% of the population may agree to being vaccinated. As a result, there is an urgent need to develop equitable and accessible interventions to address vaccine hesitancy at the population level. STUDY DESIGN: & Method: We report the development of a scalable digital intervention seeking to address COVID-19 vaccine hesitancy and enhance uptake of COVID-19 vaccines in the United Kingdom. Guided by motivational interviewing (MI) principles, the intervention includes a series of therapeutic dialogues addressing 10 key concerns of vaccine-hesitant individuals. Development of the intervention occurred linearly across four stages. During stage 1, we identified common reasons for COVID-19 vaccine hesitancy through analysis of existing survey data, a rapid systematic literature review, and public engagement workshops. Stage 2 comprised qualitative interviews with medical, immunological, and public health experts. Rapid content and thematic analysis of the data provided evidence-based responses to common vaccine concerns. Stage 3 involved the development of therapeutic dialogues through workshops with psychological and digital behaviour change experts. Dialogues were developed to address concerns using MI principles, including embracing resistance and supporting self-efficacy. Finally, stage 4 involved digitisation of the dialogues and pilot testing with members of the public. DISCUSSION: The digital intervention provides an evidence-based approach to addressing vaccine hesitancy through MI principles. The dialogues are user-selected, allowing exploration of relevant issues associated with hesitancy in a non-judgmental context. The text-based content and digital format allow for rapid modification to changing information and scalability for wider dissemination.

Is Endovascular Therapy for Stroke Cost-Effective Globally? A Systematic Review of the Literature.

OBJECTIVES: Cost-effectiveness of endovascular therapy (EVT) is a key consideration for broad use of this approach for emergent large vessel occlusion stroke. We evaluated the evidence on cost-effectiveness of EVT in comparison with best medical management from a global perspective. MATERIALS AND METHODS: This systematic review of studies published between January 2010 and May 2020 evaluated the cost effectiveness of EVT for patients with large vessel occlusion acute ischemic stroke. The gain in quality adjusted life year (QALY) and incremental cost-effectiveness ratio (ICER), expressed as cost per QALY resulting from EVT, were recorded. The study setting (country, economic perspective), decision model, and data sources used in economic models of EVT cost-effectiveness were recorded. RESULTS: Twenty-five original studies from 12 different countries were included in our review. Five of these studies were reported from a societal perspective; 18 were reported from a healthcare system perspective. Two studies used real-world data. The time horizon varied from 1 year to a lifetime; however, 18 studies reported a time horizon of >10 years. Twenty studies reported using outcome data from randomized, controlled clinical trials for their models. Nineteen studies reported using a Markov model. Incremental QALYs ranged from 0.09-3.5. All studies but 1 reported that EVT was cost-effective. CONCLUSIONS: Evidence from different countries and economic perspectives suggests that EVT for stroke treatment is cost-effective. Most cost-effectiveness studies are based on outcome data from randomized clinical trials. However, there is a need to study the cost-effectiveness of EVT based solely on real-world outcome data.

CGRP induces migraine-like symptoms in mice during both the active and inactive phases.

BACKGROUND: Circadian patterns of migraine attacks have been reported by patients but remain understudied. In animal models, circadian phases are generally not taken into consideration. In particular, rodents are nocturnal animals, yet they are most often tested during their inactive phase during the day. This study aims to test the validity of CGRP-induced behavioral changes in mice by comparing responses during the active and inactive phases. METHODS: Male and female mice of the outbred CD1 strain were administered vehicle (PBS) or CGRP (0.1 mg/kg, i.p.) to induce migraine-like symptoms. Animals were tested for activity (homecage movement and voluntary wheel running), light aversive behavior, and spontaneous pain at different times of the day and night. RESULTS: Peripheral administration of CGRP decreased the activity of mice during the first hour after administration, induced light aversive behavior, and spontaneous pain during that same period of time. Both phenotypes were observed no matter what time of the day or night they were assessed. CONCLUSIONS: A decrease in wheel activity is an additional clinically relevant phenotype observed in this model, which is reminiscent of the reduction in normal physical activity observed in migraine patients. The ability of peripheral CGRP to induce migraine-like symptoms in mice is independent of the phase of the circadian cycle. Therefore, preclinical assessment of migraine-like phenotypes can likely be done during the more convenient inactive phase of mice.

The reactive form of a C-S bond-cleaving, CO2-fixing flavoenzyme.

NADPH: 2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) is a bacterial disulfide oxidoreductase (DSOR) that, uniquely in this family, catalyzes CO2 fixation. 2-KPCC differs from other DSORs by having a phenylalanine that replaces a conserved histidine, which in typical DSORs is essential for stabilizing the reduced, reactive form of the active site. Here, using site-directed mutagenesis and stopped-flow kinetics, we examined the reactive form of 2-KPCC and its single turnover reactions with a suicide substrate and CO2 The reductive half-reaction of 2-KPCC was kinetically and spectroscopically similar to that of a typical DSOR, GSH reductase, in which the active-site histidine had been replaced with an alanine. However, the reduced, reactive form of 2-KPCC was distinct from those typical DSORs. In the absence of the histidine, the flavin and disulfide moieties were no longer coupled via a covalent or charge transfer interaction as in typical DSORs. Similar to thioredoxins, the pKa between 7.5 and 8.1 that controls reactivity appeared to be due to a single proton shared between the cysteines of the dithiol, which effectively stabilizes the attacking cysteine sulfide and renders it capable of breaking the strong C-S bond of the substrate. The lack of a histidine protected 2-KPCC's reactive intermediate from unwanted protonation; however, without its input as a catalytic acid-base, the oxidative half-reaction where carboxylation takes place was remarkably slow, limiting the overall reaction rate. We conclude that stringent regulation of protons in the DSOR active site supports C-S bond cleavage and selectivity for CO2 fixation.

Triggering the stringent response enhances synthetic methanol utilization in Escherichia coli.

Synthetic methylotrophy aims to engineer methane and methanol utilization pathways in platform hosts like Escherichia coli for industrial bioprocessing of natural gas and biogas. While recent attempts to engineer synthetic methylotrophs have proved successful, autonomous methylotrophy, i.e. the ability to utilize methane or methanol as sole carbon and energy substrates, has not yet been realized. Here, we address an important limitation of autonomous methylotrophy in E. coli: the inability of the organism to synthesize several amino acids when grown on methanol. By activating the stringent/stress response via ppGpp overproduction, or DksA and RpoS overexpression, we demonstrate improved biosynthesis of proteinogenic amino acids via endogenous upregulation of amino acid synthesis pathway genes. Thus, we were able to achieve biosynthesis of several limiting amino acids from methanol-derived carbon, in contrast to the control methylotrophic E. coli strain. This study addresses a key limitation currently preventing autonomous methylotrophy in E. coli and possibly other synthetic methylotrophs and provides insight as to how this limitation can be alleviated via stringent/stress response activation.

Engineering Escherichia coli for methanol-dependent growth on glucose for metabolite production.

Synthetic methylotrophy aims to engineer methane and methanol utilization pathways in platform hosts like Escherichia coli for industrial bioprocessing of natural gas and biogas. While recent attempts to engineer synthetic methanol auxotrophs have proved successful, these studies focused on scarce and expensive co-substrates. Here, we engineered E. coli for methanol-dependent growth on glucose, an abundant and inexpensive co-substrate, via deletion of glucose 6-phosphate isomerase (pgi), phosphogluconate dehydratase (edd), and ribose 5-phosphate isomerases (rpiAB). Since the parental strain did not exhibit methanol-dependent growth on glucose in minimal medium, we first achieved methanol-dependent growth via amino acid supplementation and used this medium to evolve the strain for methanol-dependent growth in glucose minimal medium. The evolved strain exhibited a maximum growth rate of 0.15 h-1 in glucose minimal medium with methanol, which is comparable to that of other synthetic methanol auxotrophs. Whole genome sequencing and 13C-metabolic flux analysis revealed the causative mutations in the evolved strain. A mutation in the phosphotransferase system enzyme I gene (ptsI) resulted in a reduced glucose uptake rate to maintain a one-to-one molar ratio of substrate utilization. Deletion of the e14 prophage DNA region resulted in two non-synonymous mutations in the isocitrate dehydrogenase (icd) gene, which reduced TCA cycle carbon flux to maintain the internal redox state. In high cell density glucose fed-batch fermentation, methanol-dependent acetone production resulted in 22% average carbon labeling of acetone from 13C-methanol, which far surpasses that of the previous best (2.4%) found with methylotrophic E. coli Δpgi. This study addresses the need to identify appropriate co-substrates for engineering synthetic methanol auxotrophs and provides a basis for the next steps toward industrial one-carbon bioprocessing.

Investigating relationships among cancer survivors' engagement in an online support community, social support perceptions, well-being, and moderating effects of existing (offline) social support.

PURPOSE: Socially supportive relationships help cancer survivors cope with their diagnosis and may improve quality of life; however, many survivors report unmet support and information needs. Online communities of survivors may address these needs, but research on their benefits have been equivocal. This cross-sectional, self-report study investigated relationships among cancer survivors' level of engagement in an online survivor community (The American Cancer Society Cancer Survivors Network®; CSN), perceptions of emotional/informational support available from online communities ("online social support"), well-being, and moderating effects of "offline social support." METHODS: Participants were 1255 registered users of the CSN who completed surveys between 2013 and 2014. Three types of engagement with the CSN-social/communal, interpersonal communication, and informational/search engagement-were identified through principal components analysis. Regression analyses examined hypotheses. RESULTS: More frequent social/communal and interpersonal communication engagement were associated with increased online social support (p < .0001), and the relationship between interpersonal communication engagement and online social support was strongest for survivors reporting lower offline social support (interaction ß = - .35, p < .001). Greater online social support was associated with increased well-being, but only among survivors reporting low offline social support (interaction ß = - .35, p < .0001). CONCLUSIONS: Engagement in online survivor communities may increase support perceptions that promote well-being, but benefits may accrue more to survivors reporting low offline social support. IMPLICATIONS FOR CANCER SURVIVORS: Newly diagnosed cancer survivors, particularly those with unmet emotional/informational support needs, should be given the opportunity to communicate with other survivors through online survivor support networks.

Consent for newborn screening: screening professionals' and parents' views.

OBJECTIVES: Expansion of newborn bloodspot screening (NBS) within England, which practices an informed consent model, justified examining acceptability and effectiveness of alternative consent models. STUDY DESIGN: Qualitative focus groups. METHODS: Forty-five parents and 37 screening professionals (SPs) participated. Data were analysed using thematic analysis. RESULTS: Parents and SPs initially appeared to have differing views about appropriate consent models. Most parents accepted assumed consent, if adequately informed; however, once aware of bloodspot storage, informed consent was wanted. SPs valued informed consent, but acknowledged it was difficult to obtain. Both samples wanted parents to be informed but were unclear how this could be achieved. Most parents felt NBS was not presented as optional. CONCLUSION: The simultaneous exploration of parents and SPs views, in real time is original. This rigour avoided the reliance on retrospective accounts which make it difficult to establish how decisions were made at the time. It is also unique in providing pre-interview consent models to drive the depth of data. It was rigorous in member checking. Findings suggested a preference for full disclosure of all information with some parents valuing this more than choice. Both samples queried whether current consent was sufficiently informed and voluntary. Results suggest differing tolerances of consent type if screening is solely for diagnostic purposes vs bloodspot storage. Results highlight the need for caution when examining consent model preferences without also checking knowledge, as opinions may be based on incomplete knowledge. Future research is needed to examine efficacy of proposed changes. FUNDING: National Institute for Health Research Health Technology Assessment HTAProgramme (11/62/02). TRIAL REGISTRATION: ISRCTN70227207.