Small-molecule autocatalysis drives compartment growth, competition and reproduction.

Sustained autocatalysis coupled to compartment growth and division is a key step in the origin of life, but an experimental demonstration of this phenomenon in an artificial system has previously proven elusive. We show that autocatalytic reactions within compartments-when autocatalysis, and reactant and solvent exchange outpace product exchange-drive osmosis and diffusion, resulting in compartment growth. We demonstrate, using the formose reaction compartmentalized in aqueous droplets in an emulsion, that compartment volume can more than double. Competition for a common reactant (formaldehyde) causes variation in droplet growth rate based on the composition of the surrounding droplets. These growth rate variations are partially transmitted after selective division of the largest droplets by shearing, which converts growth-rate differences into differences in droplet frequency. This shows how a combination of properties of living systems (growth, division, variation, competition, rudimentary heredity and selection) can arise from simple physical-chemical processes and may have paved the way for the emergence of evolution by natural selection.

Acceptability, reliability, and validity of a vaginal insert for the self-assessment of endometriosis-associated deep dyspareunia: a cross-sectional study.

BACKGROUND: Approximately half of people with endometriosis experience deep dyspareunia; however, there is no means of objective self-testing of endometriosis-associated deep dyspareunia. AIM: The aim of this study was to assess the acceptability, test-retest reliability, and validity of a vaginal insert for a self-assessment of endometriosis-associated deep dyspareunia. METHODS: Participants were recruited from a tertiary endometriosis center. Inclusion criteria were: 19 to 49 years of age, self-reported deep dyspareunia of ≥4 of 10, and surgically confirmed endometriosis. Participants completed 2 self-assessments using the vaginal insert to self-assess tenderness at the right and left pelvic floor, bladder, cervix-uterus, and posterior cul-de-sac (vaginal fornix). The participants recorded tenderness at each pelvic site and completed a questionnaire regarding the acceptability of the vaginal insert to assess deep dyspareunia. Test-retest reliability was assessed by correlating the tenderness scores between the 2 assessment dates. Over a 4-week period, the participants also recorded deep dyspareunia severity at each penetrative vaginal sex encounter. Validity was assessed by correlating vaginal insert tenderness to deep dyspareunia severity, and also to tenderness reported on a prior gynecologic pelvic examination. OUTCOMES: The main outcome measures were the acceptability index score, tenderness (0-10) at each pelvic site, and prospective deep dyspareunia scores (0-10) over 4 weeks. RESULTS: There were 19 participants (mean age 34 ± 7 years) who completed the study. The majority identified as female (94.7%), heterosexual (89.5%), and white (89.5%). The median acceptability index score was 0.72 (interquartile range, 0.66-0.81). For test-retest reliability, the intraclass correlation coefficients were 0.79 (P = .001) for the left pelvic floor, 0.82 (P < .001) for the right pelvic floor, 0.54 (P = .07) for the bladder, 0.89 (P < .001) for the cervix-uterus, and 0.77 (P = .003) for the cul-de-sac. The correlation between the highest self-assessed mean tenderness in each participant and self-reported deep dyspareunia over 4 weeks was r = 0.32, but correlations for each pelvic site varied significantly. Tenderness at each site on prior gynecologist pelvic exam was associated with higher self-assessed mean tenderness with the vaginal insert in each participant (effect sizes = 0.42-0.88). CLINICAL IMPLICATIONS: The vaginal insert is acceptable and reliable for the objective self-assessment of endometriosis-associated deep dyspareunia, with initial evidence of validity. STRENGTHS AND LIMITATIONS: A strength was the inclusion of participants who were avoiding sexual activity and a limitation was the small sample size. CONCLUSION: Future studies with larger sample sizes are required to further establish the validity of the vaginal insert for the self-assessment of endometriosis-associated deep dyspareunia.

Ohnut Versus a Waitlist Control for the Self-management of Endometriosis-Associated Deep Dyspareunia: Protocol for a Pilot Randomized Controlled Trial.

BACKGROUND: Endometriosis-associated deep dyspareunia is associated with reduced sexual quality of life, lower self-esteem, and impaired sexual function. OBJECTIVE: The primary objective is to assess the acceptability of a phallus length reducer (brand name: Ohnut [OhnutCo]), which is a buffer worn over the penis or a penetrating object to reduce endometriosis-associated deep dyspareunia, and the feasibility of a definitive randomized controlled trial (RCT). The secondary objective is to obtain estimates of the effectiveness of the buffer. An embedded substudy will explore the acceptability and the preliminary validity and reliability of a vaginal insert for the self-assessment of deep dyspareunia. METHODS: Ours is an investigator-initiated, 2-arm RCT. We will recruit 40 patient participants with diagnosed endometriosis between the ages of 19 and 49 years, as well as their sexual partners. The participating couples will be randomized in a 1:1 ratio into the experimental arm or the waitlist control arm. The length of the study period will be 10 weeks, during which time all participants will record deep dyspareunia severity following each episode of sexual intercourse. In weeks 1 to 4, all patient participants will record deep dyspareunia severity at each sexual encounter. In weeks 5 to 10, participants in the experimental arm will use the buffer during vaginal penetration; participants in the waitlist control arm will continue engaging in vaginal penetration as usual. Participants will complete questionnaires for assessing measures of anxiety, depression, and sexual function at baseline, at 4 weeks, and at 10 weeks. In the substudy, patient participants will self-assess dyspareunia by using a vaginal insert on 2 occasions, at least 1 week apart. The primary outcomes-the acceptability and feasibility of the buffer-will be assessed with descriptive statistics, and the secondary outcome-phallus length reducer effectiveness-will be assessed by using an analysis of covariance-based approach. For the vaginal insert, we will assess acceptability, test-retest reliability, and convergent validity via correlation analyses comparing the use of the insert to clinical examination in terms of dyspareunia assessment outcomes. RESULTS: Our pilot will provide initial data on the acceptability and effectiveness of the buffer and the feasibility of the study methodology. The results from our study are expected to be submitted for publication by the spring of 2023. As of September 2021, we have consented 31 couples into the study. CONCLUSIONS: Our study will provide preliminary evidence for the self-assessment and management of endometriosis-associated deep dyspareunia. The findings will inform the decision to proceed to a definitive RCT. TRIAL REGISTRATION: ClinicalTrials.gov NCT04370444; https://clinicaltrials.gov/ct2/show/NCT04370444. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/39834.

Noncovalent hyaluronan crosslinking by TSG-6: Modulation by heparin, heparan sulfate, and PRG4.

The size, conformation, and organization of the glycosaminoglycan hyaluronan (HA) affect its interactions with soluble and cell surface-bound proteins. HA that is induced to form stable networks has unique biological properties relative to unmodified soluble HA. AlphaLISA assay technology offers a facile and general experimental approach to assay protein-mediated networking of HA in solution. Connections formed between two end-biotinylated 50 kDa HA (bHA) chains can be detected by signal arising from streptavidin-coated donor and acceptor beads being brought into close proximity when the bHA chains are bridged by proteins. We observed that incubation of bHA with the protein TSG-6 (tumor necrosis factor alpha stimulated gene/protein 6, TNFAIP/TSG-6) leads to dimerization or higher order multimerization of HA chains in solution. We compared two different heparin (HP) samples and two heparan sulfate (HS) samples for the ability to disrupt HA crosslinking by TSG-6. Both HP samples had approximately three sulfates per disaccharide, and both were effective in inhibiting HA crosslinking by TSG-6. HS with a relatively high degree of sulfation (1.75 per disaccharide) also inhibited TSG-6 mediated HA networking, while HS with a lower degree of sulfation (0.75 per disaccharide) was less effective. We further identified Proteoglycan 4 (PRG4, lubricin) as a TSG-6 ligand, and found it to inhibit TSG-6-mediated HA crosslinking. The effects of HP, HS, and PRG4 on HA crosslinking by TSG-6 were shown to be due to HP/HS/PRG4 inhibition of HA binding to the Link domain of TSG-6. Using the AlphaLISA platform, we also tested other HA-binding proteins for ability to create HA networks. The G1 domain of versican (VG1) effectively networked bHA in solution but required a higher concentration than TSG-6. Cartilage link protein (HAPLN1) and the HA binding protein segment of aggrecan (HABP, G1-IGD-G2) showed only low and variable magnitude HA networking effects. This study unambiguously demonstrates HA crosslinking in solution by TSG-6 and VG1 proteins, and establishes PRG4, HP and highly sulfated HS as modulators of TSG-6 mediated HA crosslinking.

Selective isolation of hyaluronan by solid phase adsorption to silica.

A solid phase adsorption method for selective isolation of hyaluronan (HA) from biological samples is presented. Following enzymatic degradation of protein, HA can be separated from sulfated glycosaminoglycans, other unsulfated glycosaminoglycans, nucleic acids, and proteolytic fragments by adsorption to amorphous silica at specific salt concentrations. The adsorbed HA can be released from silica using neutral and basic aqueous solutions. HA ranging in size from ∼9 kDa to MDa polymers has been purified by this method from human serum and conditioned medium of cultured cells.

Intracranial injuries on computed tomography head scans in infants investigated for suspected physical abuse: a retrospective review.

BACKGROUND: UK national guidelines recommend that investigation of infants (aged <12 months) with suspected physical abuse should always include CT head scans. Such imaging carries small but recognised risks from radiation exposure. Studies report a range of yields for occult intracranial injuries in suspected physical abuse. AIMS: To report the yield of intracranial injuries on CT head scans carried out for suspected physical abuse in infants, compare yields for those presenting with or without signs of head injury and to describe selected clinical and radiological features. METHODS: A retrospective cross-sectional review of case records of infants undergoing skeletal survey for suspected physical abuse in Wessex, England. The main outcome measure was yield of intracranial injuries on CT head scan. RESULTS: In total, n=363 CT head scans were included (n=275 aged <6 months). The overall yield of intracranial injury was 37 (10%). Among 68 infants presenting with neurological signs or skull fractures, yield was 36 (53%) compared with just 1 (0.34%) of 295 without neurological signs or skull fractures. This one intracranial injury was found to be consistent with an accidental fall. Scalp injury was the only additional clinical feature associated with intracranial injury. CONCLUSION: In suspected physical abuse, CT head scans should be carried out in infants who present with neurological signs, skull fractures or scalp injuries. However, in balancing potential risks and benefits, we question the value of performing a CT head scan in every infant investigated for suspected physical abuse.

Environmental control programs the emergence of distinct functional ensembles from unconstrained chemical reactions.

Many approaches to the origin of life focus on how the molecules found in biology might be made in the absence of biological processes, from the simplest plausible starting materials. Another approach could be to view the emergence of the chemistry of biology as process whereby the environment effectively directs "primordial soups" toward structure, function, and genetic systems over time. This does not require the molecules found in biology today to be made initially, and leads to the hypothesis that environment can direct chemical soups toward order, and eventually living systems. Herein, we show how unconstrained condensation reactions can be steered by changes in the reaction environment, such as order of reactant addition, and addition of salts or minerals. Using omics techniques to survey the resulting chemical ensembles we demonstrate there are distinct, significant, and reproducible differences between the product mixtures. Furthermore, we observe that these differences in composition have consequences, manifested in clearly different structural and functional properties. We demonstrate that simple variations in environmental parameters lead to differentiation of distinct chemical ensembles from both amino acid mixtures and a primordial soup model. We show that the synthetic complexity emerging from such unconstrained reactions is not as intractable as often suggested, when viewed through a chemically agnostic lens. An open approach to complexity can generate compositional, structural, and functional diversity from fixed sets of simple starting materials, suggesting that differentiation of chemical ensembles can occur in the wider environment without the need for biological machinery.

Approach to classify, separate, and enrich objects in groups using ensemble sorting.

The sorting of objects into groups is a fundamental operation, critical in the preparation and purification of populations of cells, crystals, beads, or droplets, necessary for research and applications in biology, chemistry, and materials science. Most of the efforts exploring such purification have focused on two areas: the degree of separation and the measurement precision required for effective separation. Conventionally, achieving good separation ultimately requires that the objects are considered one by one (which can be both slow and expensive), and the ability to measure the sorted objects by increasing sensitivity as well as reducing sorting errors. Here we present an approach to sorting that addresses both critical limitations with a scheme that allows us to approach the theoretical limit for the accuracy of sorting decisions. Rather than sorting individual objects, we sort the objects in ensembles, via a set of registers which are then in turn sorted themselves into a second symmetric set of registers in a lossless manner. By repeating this process, we can arrive at high sorting purity with a low set of constraints. We demonstrate both the theory behind this idea and identify the critical parameters (ensemble population and sorting time), and show the utility and robustness of our method with simulations and experimental systems spanning several orders of scale, sorting populations of macroscopic beads and microfluidic droplets. Our method is general in nature and simplifies the sorting process, and thus stands to enhance many different areas of science, such as purification, enrichment of rare objects, and separation of dynamic populations.

A recursive microfluidic platform to explore the emergence of chemical evolution.

We propose that a chemically agnostic approach to explore the origin of life, using an automated recursive platform based on droplet microfluidics, could be used to induce artificial chemical evolution by iterations of growth, speciation, selection, and propagation. To explore this, we set about designing an open source prototype of a fully automated evolution machine, comprising seven modules. These modules are a droplet generator, droplet transfer, passive and active size sorting, splitter, incubation chamber, reservoir, and injectors, all run together via a LabVIEWTM program integration system. Together we aim for the system to be used to drive cycles of droplet birth, selection, fusion, and propagation. As a proof of principle, in addition to the working individual modules, we present data showing the osmotic exchange of glycylglycine containing and pure aqueous droplets, showing that the fittest droplets exhibit higher osomolarity relative to their neighbours, and increase in size compared to their neighbours. This demonstrates the ability of our platform to explore some different physicochemical conditions, combining the efficiency and unbiased nature of automation with our ability to select droplets as functional units based on simple criteria.

Chromophobe renal cell carcinoma recurrence in the ureter: A late presentation of a rare metastasis.

We describe a case of recurrence of chromophobe renal cell carcinoma 8 years after successful surgical treatment of primary localized disease in the left kidney. The primary tumour had exhibited neither gross nor histological evidence of lymphovascular infiltration. The recurrence occurred in the residual left ureteric stump - a finding that, to the best of our knowledge, has not previously been reported.

Varicocele.

INTRODUCTION: Varicocele is estimated to affect about 15% of the general male population. It usually occurs only on the left side, and is often asymptomatic. There is little evidence that varicocele reduces male fertility, although it is found in 12% of male partners of couples presenting with infertility and in 25% of men with abnormal semen analysis. METHODS AND OUTCOMES: We conducted a systematic overview, aiming to answer the following clinical question: What are the effects of treatments in adult males with varicocele? We searched: Medline, Embase, The Cochrane Library, and other important databases up to November 2013 (Clinical Evidence overviews are updated periodically; please check our website for the most up-to-date version of this overview). RESULTS: Searching of electronic databases retrieved 203 studies. After deduplication and removal of conference abstracts, 91 records were screened for inclusion in the overview. Appraisal of titles and abstracts led to the exclusion of 53 studies and the further review of 38 full publications. Of the 38 full articles evaluated, one existing systematic review was updated and two systematic reviews and five RCTs were added at this update. We performed a GRADE evaluation of nine PICO combinations. CONCLUSIONS: In this systematic overview we categorised the efficacy for four interventions, based on information relating to the effectiveness of embolisation, expectant management, sclerotherapy, and surgical ligation.

Innovations in Practice: 'Off-label' clonidine: UK Paediatric and Child and Adolescent Psychiatry prescribing practice for sleep problems.

BACKGROUND: Psychopharmacological prescribing of clonidine has been described in Australia and in the United States but not in Europe. This study explores UK clinician experience of clonidine 'off label' in treating paediatric sleep problems in the context of a paucity of evidence for its use. METHODS: Survey of UK Child and Adolescent Psychiatrists (CAPs) and specialist Paediatricians. RESULTS: Of 389 respondents (30% Paediatricians, 70% CAPs), 172 prescribed clonidine and 85 having used it for treating sleep. Treatment targets were sleep onset, night waking and nonspecific sleep problems, and carer respite, in patients with significant coexisting neurological and neurodevelopmental disorders. Most used clonidine as a second line medication after trying nonpharmacological approaches. The majority reported initial effectiveness, and adverse effects were mild or transient. Issues of reducing long-term effectiveness, drug tolerance and considerable differences in dosing regimes were identified. CONCLUSIONS: Clonidine use appears less widespread in the United Kingdom than reported elsewhere. It is seen as a potentially effective and safe intervention but this study highlights the need for good quality RCT evidence for the most effective use of clonidine in paediatric sleep disorders.

Comprehensive experimental fitness landscape and evolutionary network for small RNA.

The origin of life is believed to have progressed through an RNA world, in which RNA acted as both genetic material and functional molecules. The structure of the evolutionary fitness landscape of RNA would determine natural selection for the first functional sequences. Fitness landscapes are the subject of much speculation, but their structure is essentially unknown. Here we describe a comprehensive map of a fitness landscape, exploring nearly all of sequence space, for short RNAs surviving selection in vitro. With the exception of a small evolutionary network, we find that fitness peaks are largely isolated from one another, highlighting the importance of historical contingency and indicating that natural selection would be constrained to local exploration in the RNA world.

Cascade of reduced speed and accuracy after errors in enzyme-free copying of nucleic acid sequences.

Nonenzymatic, template-directed synthesis of nucleic acids is a paradigm for self-replicating systems. The evolutionary dynamics of such systems depend on several factors, including the mutation rates, relative replication rates, and sequence characteristics of mutant sequences. We measured the kinetics of correct and incorrect monomer insertion downstream of a primer-template mismatch (mutation), using a range of backbone structures (RNA, DNA, and LNA templates and RNA and DNA primers) and two types of 5'-activated nucleotides (oxyazabenzotriazolides and imidazolides, i.e., nucleoside 5'-phosphorimidazolides). Our study indicated that for all systems studied, an initial mismatch was likely to be followed by another error (54-75% of the time), and extension after a single mismatch was generally 10-100 times slower than extension without errors. If the mismatch was followed by a matched base pair, the extension rate recovered to nearly normal levels. On the basis of these data, we simulated nucleic acid replication in silico, which indicated that a primer suffering an initial error would lag behind properly extended counterparts due to a cascade of subsequent errors and kinetic stalling, with the typical mutational event consisting of several consecutive errors. Our study also included different sequence contexts, which suggest the presence of cooperativity among monomers affecting both absolute rate (by up to 2 orders of magnitude) and fidelity. The results suggest that molecular evolution in enzyme-free replication systems would be characterized by large "leaps" through sequence space rather than isolated point mutations, perhaps enabling rapid exploration of diverse sequences. The findings may also be useful for designing self-replicating systems combining high fidelity with evolvability.

The plausibility of RNA-templated peptides: simultaneous RNA affinity for adjacent peptide side chains.

According to the RNA world hypothesis, coded peptide synthesis (translation) must have been first catalyzed by RNAs. Here, we show that small RNA sequences can simultaneously bind the dissimilar amino acids His and Phe in peptide linkage. We used in vitro counterselection/selection to isolate a pool of RNAs that bind the dipeptide NH(2)-His-Phe-COOH with K (D) ranging from 36 to 480 µM. These sites contact both side chains, usually including the protonated imidazole of His, but bind-free L: -His and L: -Phe with much lower, sometimes undetectable, affinities. The most frequent His-Phe sites do not usually contain previously isolated sites for individual amino acids, and are only ≈35 % larger than previously known separate His and Phe sites. Nonetheless, His-Phe sites appear enriched in His anticodons, as previous L: -His sites also were. Accordingly, these data add to existing experimental evidence for a stereochemical genetic code. In these peptide sites, bound amino acids approach each other to a proximity that allows a covalent peptide linkage. Isolation of several RNAs embracing two amino acids with a linking peptide bond supports the idea that a direct-RNA-template could encode primordial peptides, though crucial experiments remain.

Phototrophic phylotypes dominate mesothermal microbial mats associated with hot springs in Yellowstone National Park.

The mesothermal outflow zones (50-65°C) of geothermal springs often support an extensive zone of green and orange laminated microbial mats. In order to identify and compare the microbial inhabitants of morphologically similar green-orange mats from chemically and geographically distinct springs, we generated and analyzed small-subunit ribosomal RNA (rRNA) gene amplicons from six mesothermal mats (four previously unexamined) in Yellowstone National Park. Between three and six bacterial phyla dominated each mat. While many sequences bear the highest identity to previously isolated phototrophic genera belonging to the Cyanobacteria, Chloroflexi, and Chlorobi phyla, there is also frequent representation of uncultured, unclassified members of these groups. Some genus-level representatives of these dominant phyla were found in all mats, while others were unique to a single mat. Other groups detected at high frequencies include candidate divisions (such as the OP candidate clades) with no cultured representatives or complete genomes available. In addition, rRNA genes related to the recently isolated and characterized photosynthetic acidobacterium "Candidatus Chloracidobacterium thermophilum" were detected in most mats. In contrast to microbial mats from well-studied hypersaline environments, the mesothermal mats in this study accrue less biomass and are substantially less diverse, but have a higher proportion of known phototrophic organisms. This study provides sequences appropriate for accurate phylogenetic classification and expands the molecular phylogenetic survey of Yellowstone microbial mats.

Treatment of severe steroid-dependent nephrotic syndrome (SDNS) in children with tacrolimus.

BACKGROUND: Severe steroid-dependent nephrotic syndrome (SDNS) is a common type of nephrotic syndrome (NS) observed in childhood. Steroid-sparing agents such as calcineurin inhibitors (CNIs) are used to avoid steroid toxicity in SDNS. Tacrolimus (TAC) has been prescribed for maintaining remission of NS in patients who have developed treatment resistance or adverse effects with cyclosporin A (CYA) at our institution since 1995. The aim of this study was to compare the efficacy and complications of TAC with CYA in the management of severe SDNS. METHODS: We report a retrospective longitudinal clinical series of patients with SDNS, all of whom have been treated with TAC. RESULTS: Ten SDNS children (eight males) were reviewed quarterly from time of initial referral to the present day during 93 completed treatment patient years. Nine patients had minimal change disease and one had focal segmental glomerulosclerosis on their first biopsy. The median age at diagnosis was 2.9 years (range 1.6-12.9). The median age at initial referral was 3.9 years (range 2.2-12.9). All patients initially responded to prednisolone at 60 mg/m2/day, and subsequent frequent relapses were treated sequentially with oral cyclophosphamide 168 mg/kg over 8-12 weeks (n = 10), CYA (n = 10), intravenous mustine or a second course of cyclophosphamide (n = 7) and then TAC (n = 10). The initial daily treatment of CYA and TAC in two divided doses was 5 and 0.1 mg/kg/day, respectively; targeted 12 h blood drug levels were of 50-100 microg/l for CYA and 5-10 microg/l for TAC. Patients underwent renal biopsy and the formal glomerular filtration rate (GFR) was measured using plasma clearance of the Inutest method every 2-3 years while receiving CNIs. Six patients continued with TAC; in four patients, TAC was discontinued because of poor response (n = 2), hypertension (n = 1) and glucose intolerance (n = 1). For CYA and TAC treatment periods, the median NS relapse rate was two and one relapses per year, respectively, and cumulative steroid dosage was 73.9 and 105.2 mg/kg/day, respectively (P = 0.54). The reduction in GFR was 5.8 and 11.7 ml/min/1.73 m2 during these periods. Three of the 10 patients showed histological evidence of mild CNI nephrotoxicity over the whole of the CNI treatment period despite achieving target therapeutic drug levels; no significant change in measured or calculated GFR over this prolonged CNI therapy was observed. Antihypertensive medication was prescribed for 11 of 31 CYA and 22 of 40 TAC treatment years. Growth was maintained during the entire CNI therapy period with median change in height SD scores (SDS) of +0.37 and -0.03 over CYA and TAC, respectively (P = 0.13). CONCLUSION: In conclusion, we observed that the replacement of CYA by TAC does not lead to a better management of severe SDNS.