MHC-Fine: Fine-tuned AlphaFold for precise MHC-peptide complex prediction.

The precise prediction of major histocompatibility complex (MHC)-peptide complex structures is pivotal for understanding cellular immune responses and advancing vaccine design. In this study, we enhanced AlphaFold's capabilities by fine-tuning it with a specialized dataset consisting of exclusively high-resolution class I MHC-peptide crystal structures. This tailored approach aimed to address the generalist nature of AlphaFold's original training, which, while broad-ranging, lacked the granularity necessary for the high-precision demands of class I MHC-peptide interaction prediction. A comparative analysis was conducted against the homology-modeling-based method Pandora as well as the AlphaFold multimer model. Our results demonstrate that our fine-tuned model outperforms others in terms of root-mean-square deviation (median value for Cα atoms for peptides is 0.66 Å) and also provides enhanced predicted local distance difference test scores, offering a more reliable assessment of the predicted structures. These advances have substantial implications for computational immunology, potentially accelerating the development of novel therapeutics and vaccines by providing a more precise computational lens through which to view MHC-peptide interactions.

Expanding FTMap for Fragment-Based Identification of Pharmacophore Regions in Ligand Binding Sites.

The knowledge of ligand binding hot spots and of the important interactions within such hot spots is crucial for the design of lead compounds in the early stages of structure-based drug discovery. The computational solvent mapping server FTMap can reliably identify binding hot spots as consensus clusters, free energy minima that bind a variety of organic probe molecules. However, in its current implementation, FTMap provides limited information on regions within the hot spots that tend to interact with specific pharmacophoric features of potential ligands. E-FTMap is a new server that expands on the original FTMap protocol. E-FTMap uses 119 organic probes, rather than the 16 in the original FTMap, to exhaustively map binding sites, and identifies pharmacophore features as atomic consensus sites where similar chemical groups bind. We validate E-FTMap against a set of 109 experimentally derived structures of fragment-lead pairs, finding that highly ranked pharmacophore features overlap with the corresponding atoms in both fragments and lead compounds. Additionally, comparisons of mapping results to ensembles of bound ligands reveal that pharmacophores generated with E-FTMap tend to sample highly conserved protein-ligand interactions. E-FTMap is available as a web server at https://eftmap.bu.edu.

Mapping of antibody epitopes based on docking and homology modeling.

Antibodies are key proteins produced by the immune system to target pathogen proteins termed antigens via specific binding to surface regions called epitopes. Given an antigen and the sequence of an antibody the knowledge of the epitope is critical for the discovery and development of antibody based therapeutics. In this work, we present a computational protocol that uses template-based modeling and docking to predict epitope residues. This protocol is implemented in three major steps. First, a template-based modeling approach is used to build the antibody structures. We tested several options, including generation of models using AlphaFold2. Second, each antibody model is docked to the antigen using the fast Fourier transform (FFT) based docking program PIPER. Attention is given to optimally selecting the docking energy parameters depending on the input data. In particular, the van der Waals energy terms are reduced for modeled antibodies relative to x-ray structures. Finally, ranking of antigen surface residues is produced. The ranking relies on the docking results, that is, how often the residue appears in the docking poses' interface, and also on the energy favorability of the docking pose in question. The method, called PIPER-Map, has been tested on a widely used antibody-antigen docking benchmark. The results show that PIPER-Map improves upon the existing epitope prediction methods. An interesting observation is that epitope prediction accuracy starting from antibody sequence alone does not significantly differ from that of starting from unbound (i.e., separately crystallized) antibody structure.

Comet Assay Profiling of FLASH-Induced Damage: Mechanistic Insights into the Effects of FLASH Irradiation.

Numerous studies have demonstrated the normal tissue-sparing effects of ultra-high dose rate 'FLASH' irradiation in vivo, with an associated reduction in damage burden being reported in vitro. Towards this, two key radiochemical mechanisms have been proposed: radical-radical recombination (RRR) and transient oxygen depletion (TOD), with both being proposed to lead to reduced levels of induced damage. Previously, we reported that FLASH induces lower levels of DNA strand break damage in whole-blood peripheral blood lymphocytes (WB-PBL) ex vivo, but our study failed to distinguish the mechanism(s) involved. A potential outcome of RRR is the formation of crosslink damage (particularly, if any organic radicals recombine), whilst a possible outcome of TOD is a more anoxic profile of induced damage resulting from FLASH. Therefore, the aim of the current study was to profile FLASH-induced damage via the Comet assay, assessing any DNA crosslink formation as a putative marker of RRR and/or anoxic DNA damage formation as an indicative marker of TOD, to determine the extent to which either mechanism contributes to the "FLASH effect". Following FLASH irradiation, we see no evidence of any crosslink formation; however, FLASH irradiation induces a more anoxic profile of induced damage, supporting the TOD mechanism. Furthermore, treatment of WB-PBLs pre-irradiation with BSO abrogates the reduced strand break damage burden mediated by FLASH exposures. In summary, we do not see any experimental evidence to support the RRR mechanism contributing to the reduced damage burden induced by FLASH. However, the observation of a greater anoxic profile of damage following FLASH irradiation, together with the BSO abrogation of the reduced strand break damage burden mediated by FLASH, lends further support to TOD being a driver of the reduced damage burden plus a change in the damage profile mediated by FLASH.

The First Thousand Days of Life.

The first thousand days of life, composing the 270 days of pregnancy and the first two years (730 days) of life, is at once a critical and vulnerable time for human development. It is a time in which the human person is to a large extent "embodied," becoming the integrated mind-brain-body-spirit that defines every human being. This embodiment is set in motion at fertilization and continues with the unfolding development of the embryo. By six weeks in utero, the brain is forming via ongoing neurogenesis, neuronal migration, synaptogenesis, and myelination. By the age of two the brain will be 80 percent of its adult size. As the brain develops, it connects to other developing body systems including the immune, endocrine, metabolic, gastrointestinal, cardiovascular and musculoskeletal systems. In this process of interconnection, a human being is shaped by both internal and external environments, both at the stage of the fetus and the stage of the infant. Human well-being and flourishing depends upon making these first thousand days as safe, secure, and healthy as possible. Physician practices, local health policy, and global health advocacy should focus on optimizing the first thousand days. This should include pre-conception care, pregnancy, safe birth, infant nutrition and fostering secure emotional and relational attachments.

Postoperative biliary anastomotic strictures after pancreaticoduodenectomy.

BACKGROUND: Biliary anastomotic stricture (BAS) is an uncommon complication of pancreaticoduodenectomy (PD). As PDs are performed more frequently, BAS may become a more common pathologic entity requiring clinical engagement. The aim of this study was to report the incidence of BAS in the modern era of pancreatic surgery and identify risk factors associated with it. METHODS: Patients undergoing PD at the Johns Hopkins Hospital between 2007 and 2016 were identified using an institutional registry and clinicopathological features were analyzed to identify risk factors associated with BAS. RESULTS: Of 2125 patients identified, 103 (4.9%) developed BAS. Factors independently associated with BAS included laparoscopic approach (HR:2.83,95%CI:1.35-5.92, p = 0.006), postoperative pancreatic fistula (HR:2.45,95%CI:1.56-4.16,p < 0.001), postoperative bile leak (BL) (HR:5.26,95%CI:2.45-11.28,p < 0.001), and administration of adjuvant radiation therapy (HR:6.01,95%CI:3.19-11.34,p < 0.001). Malignant pathology was associated with lower rates of BAS (HR:0.52,95%CI:0.30-0.92, p = 0.025). BL was associated with higher rates of early-BAS (HR:16.49,95%CI:3.28-82.94, p = 0.001) while use of Vicryl suture for biliary enteric anastomosis was associated with lower rates of early-BAS (HR:0.20,95%CI:0.05-0.93, p = 0.041). CONCLUSION: Approximately 5% of patients undergoing PD experience BAS. Multiple factors are associated with the development and timing of BAS.

A Diagnostic Stewardship Intervention To Improve Blood Culture Use among Adult Nonneutropenic Inpatients: the DISTRIBUTE Study.

Interventions to optimize blood culture (BCx) practices in adult inpatients are limited. We conducted a before-after study evaluating the impact of a diagnostic stewardship program that aimed to optimize BCx use in a medical intensive care unit (MICU) and five medicine units at a large academic center. The program included implementation of an evidence-based algorithm detailing indications for BCx use and education and feedback to providers about BCx rates and indication inappropriateness. Neutropenic patients were excluded. BCx rates from contemporary control units were obtained for comparison. The primary outcome was the change in BCxs ordered with the intervention. Secondary outcomes included proportion of inappropriate BCx, solitary BCx, and positive BCx. Balancing metrics included compliance with the Centers for Medicare and Medicaid Services (CMS) SEP-1 BCx component, 30-day readmission, and all-cause in-hospital and 30-day mortality. After the intervention, BCx rates decreased from 27.7 to 22.8 BCx/100 patient-days (PDs) in the MICU (P = 0.001) and from 10.9 to 7.7 BCx/100 PD for the 5 medicine units combined (P < 0.001). BCx rates in the control units did not decrease significantly (surgical intensive care unit [ICU], P = 0.06; surgical units, P = 0.15). The proportion of inappropriate BCxs did not significantly change with the intervention (30% in the MICU and 50% in medicine units). BCx positivity increased in the MICU (from 8% to 11%, P < 0.001). Solitary BCxs decreased by 21% in the medicine units (P < 0.001). Balancing metrics were similar before and after the intervention. BCx use can be optimized with clinician education and practice guidance without affecting sepsis quality metrics or mortality.

A multicentre audit to assess the effectiveness of the British Orthodontic Society 'Hold that Smile' retainer videos.

INTRODUCTION: Retention is a crucial part of orthodontic treatment; however, patients often do not wear their retainers as advised. The British Orthodontic Society developed the 'Hold that Smile' campaign in 2017, to improve patient knowledge about retention. Information is provided in two formats: a cartoon and a conventional film. OBJECTIVE: To assess whether patients find the 'Hold that Smile' videos useful and whether they improved patients' intended retainer wear. The gold standard was that 90% of patients should intend to wear their retainers in the long term after watching the videos. DESIGN: National multicentre audit. SETTING: Nine units in the UK. METHODS: Patients aged ⩾ 10 years, in fixed appliances or retention, watched the retainer videos and then completed a questionnaire that was designed specifically for this audit. Each unit collected data for approximately 30 patients. RESULTS: Data were collected for 278 patients in total. The average age was 17.9 years; 64.4% of patients were female and 35.6% were male. Most patients (86.3%) watched both videos and, of these, 44.1% preferred the film, 31.3% preferred the cartoon and 24.6% had no preference. The majority of patients (81.3%) felt that the film provided them with new information, compared with a lower percentage (48.5%) for the cartoon. More patients said they would recommend the film (76.3%) compared with the cartoon (63.3%). Before watching the videos, 77.0% of patients felt they knew about long-term retainer wear and 74.3% of those intended to wear their retainers in the long term. After watching the videos, 96.4% of all patients thought they would now wear their retainers long term. CONCLUSION: After watching the videos, there was a notable increase in the number of patients planning to wear their retainers long term and the gold standard was met. Therefore, these videos may be beneficial in improving understanding and compliance with retention.

Phylogeny and Evolution of RNA 3'-Nucleotidyltransferases in Bacteria.

The tRNA nucleotidyltransferases and poly(A) polymerases belong to a superfamily of nucleotidyltransferases. The amino acid sequences of a number of bacterial tRNA nucleotidyltransferases and poly(A) polymerases have been used to construct a rooted, neighbor-joining phylogenetic tree. Using information gleaned from that analysis, along with data from the rRNA-based phylogenetic tree, structural data available on a number of members of the superfamily and other biochemical information on the superfamily, it is possible to suggest a scheme for the evolution of the bacterial tRNA nucleotidyltransferases and poly(A) polymerases from ancestral species. Elements of that scheme are discussed along with questions arising from the scheme which can be explored experimentally.

Proteome analysis of biofilm produced by a Fusarium falciforme keratitis infectious agent.

Biofilms are structures that confer adaptive ability to and facilitate the virulence of fungal pathogens. Certain multi-functional proteins have been shown to be involved in fungal pathogenesis and these proteins may also be implicated in biofilm formation. The aim of this study was to identify a fungal agent isolated from the human cornea, to analyze the ability of this organism to form biofilms in vitro and to investigate protein expression in this condition. The fungus was identified by phylogenetic inference analysis. Biofilm formation and structure were evaluated by colorimetric methods and by optical and electron microscopy. We also resolved proteins obtained from biofilms and planktonic cultures by two-dimensional gel electrophoresis and identified those proteins by mass spectrometry. The fungus was identified as Fusarium falciforme. Colorimetric analysis and microscopy revealed that the highest level of biofilm formation was obtained at a concentration of 1 × 106 conidia/mL with 96 h of incubation at 28 °C. The biofilm architecture consisted of an extracellular matrix that embedded fungal filaments. We found nineteen proteins that were over-expressed in biofilms, as compared with planktonic cultures, and six proteins with unique expression in biofilms. Among the more abundant proteins identified were: transketolase, a putative antigen 1, enolase, phosphoglycerate kinase and ATP-citrate synthase. Some of these proteins are involved in basal metabolism, function as multi-functional proteins or have been described as potential virulence factors. We focused on the expression in biofilm of the enzyme, enolase, which was determined by real-time PCR. Our findings provide a perspective on the proteins associated with the formation of biofilms in vitro by an F. falciforme keratitis isolate.

Identification and biofilm development by a new fungal keratitis aetiologic agent.

BACKGROUND: In recent years, human keratitis caused by fungal plant pathogens has become more common. Biofilm is a structure that confers adaptations and virulence to fungi in keratitis. Neoscytalidium spp. are phytopathogenic and recently have been recognised as a human pathogen, using biofilm formation as a virulence factor. OBJECTIVES: The aim of this study was isolation, identification (at the species level) and characterisation of a new fungal keratitis agent. PATIENTS/METHODS: The fungus was isolated from a 67-year-old male patient with a corneal ulcer. Biofilm formation and structure were evaluated by colorimetric methods and microscopy. To identify the fungus, morphological characteristics were examined and a phylogenetic analysis was performed. RESULTS AND CONCLUSIONS: We report the identification of a fungus, a member of the genus Neoscytalidium which is associated with human keratitis. Phylogenetic analysis and morphological observations on conidiogenous cells, which occur only in arthric chains in aerial mycelium and the coelomycetous synasexual morph is absent, identified a new species, Neoscytalidium oculus sp. nov. The fungus formed biofilm at a concentration of 1 × 106  conidia/mL, during 96 hours of incubation at 37°C, and also manifested haemolysis and melanin production. This is the first report in Latin America of a new species of Neoscytalidium from a clinical isolate has been identified.

Genome-Wide Adductomics Analysis Reveals Heterogeneity in the Induction and Loss of Cyclobutane Thymine Dimers across Both the Nuclear and Mitochondrial Genomes.

The distribution of DNA damage and repair is considered to occur heterogeneously across the genome. However, commonly available techniques, such as the alkaline comet assay or HPLC-MS/MS, measure global genome levels of DNA damage, and do not reflect potentially significant events occurring at the gene/sequence-specific level, in the nuclear or mitochondrial genomes. We developed a method, which comprises a combination of Damaged DNA Immunoprecipitation and next generation sequencing (DDIP-seq), to assess the induction and repair of DNA damage induced by 0.1 J/cm2 solar-simulated radiation at the sequence-specific level, across both the entire nuclear and mitochondrial genomes. DDIP-seq generated a genome-wide, high-resolution map of cyclobutane thymine dimer (T<>T) location and intensity. In addition to being a straightforward approach, our results demonstrated a clear differential distribution of T<>T induction and loss, across both the nuclear and mitochondrial genomes. For nuclear DNA, this differential distribution existed at both the sequence and chromosome level. Levels of T<>T were much higher in the mitochondrial DNA, compared to nuclear DNA, and decreased with time, confirmed by qPCR, despite no reported mechanisms for their repair in this organelle. These data indicate the existence of regions of sensitivity and resistance to damage formation, together with regions that are fully repaired, and those for which > 90% of damage remains, after 24 h. This approach offers a simple, yet more detailed approach to studying cellular DNA damage and repair, which will aid our understanding of the link between DNA damage and disease.

MTH1 deficiency selectively increases non-cytotoxic oxidative DNA damage in lung cancer cells: more bad news than good?

BACKGROUND: Targeted therapies are based on exploiting cancer-cell-specific genetic features or phenotypic traits to selectively kill cancer cells while leaving normal cells unaffected. Oxidative stress is a cancer hallmark phenotype. Given that free nucleotide pools are particularly vulnerable to oxidation, the nucleotide pool sanitising enzyme, MTH1, is potentially conditionally essential in cancer cells. However, findings from previous MTH1 studies have been contradictory, meaning the relevance of MTH1 in cancer is still to be determined. Here we ascertained the role of MTH1 specifically in lung cancer cell maintenance, and the potential of MTH1 inhibition as a targeted therapy strategy to improve lung cancer treatments. METHODS: Using siRNA-mediated knockdown or small-molecule inhibition, we tested the genotoxic and cytotoxic effects of MTH1 deficiency on H23 (p53-mutated), H522 (p53-mutated) and A549 (wildtype p53) non-small cell lung cancer cell lines relative to normal MRC-5 lung fibroblasts. We also assessed if MTH1 inhibition augments current therapies. RESULTS: MTH1 knockdown increased levels of oxidatively damaged DNA and DNA damage signaling alterations in all lung cancer cell lines but not normal fibroblasts, despite no detectable differences in reactive oxygen species levels between any cell lines. Furthermore, MTH1 knockdown reduced H23 cell proliferation. However, unexpectedly, it did not induce apoptosis in any cell line or enhance the effects of gemcitabine, cisplatin or radiation in combination treatments. Contrastingly, TH287 and TH588 MTH1 inhibitors induced apoptosis in H23 and H522 cells, but only increased oxidative DNA damage levels in H23, indicating that they kill cells independently of DNA oxidation and seemingly via MTH1-distinct mechanisms. CONCLUSIONS: MTH1 has a NSCLC-specific p53-independent role for suppressing DNA oxidation and genomic instability, though surprisingly the basis of this may not be reactive-oxygen-species-associated oxidative stress. Despite this, overall our cell viability data indicates that targeting MTH1 will likely not be an across-the-board effective NSCLC therapeutic strategy; rather it induces non-cytotoxic DNA damage that could promote cancer heterogeneity and evolution.

Graphene Devices for Tabletop and High-Current Quantized Hall Resistance Standards.

We report the performance of a quantum Hall resistance standard based on epitaxial graphene maintained in a 5-T tabletop cryocooler system. This quantum resistance standard requires no liquid helium and can operate continuously, allowing year-round accessibility to quantized Hall resistance measurements. The ν = 2 plateau, with a value of R K/2, also seen as R H, is used to scale to 1 kΩ using a binary cryogenic current comparator (BCCC) bridge and a direct current comparator (DCC) bridge. The uncertainties achieved with the BCCC are such as those obtained in the state-of-the-art measurements using GaAs-based devices. BCCC scaling methods can achieve large resistance ratios of 100 or more, and while room temperature DCC bridges have smaller ratios and lower current sensitivity, they can still provide alternate resistance scaling paths without the need for cryogens and superconducting electronics. Estimates of the relative uncertainties of the possible scaling methods are provided in this report, along with a discussion of the advantages of several scaling paths. The tabletop system limits are addressed as are potential solutions for using graphene standards at higher currents.

Glass ionomer or composite resin for primary molars.

Data sourcesPubMed, Scopus, Web of Science, Virtual Health Library (VHL), Cochrane Library, Clinical Trials and OpenGrey.Study selectionRandomised controlled trials comparing the clinical effectiveness of Class II restorations performed with conventional (C-GIC) or resin-modified glass ionomer cement (RM-GIC) and composite resin (CR) in primary molar teeth. No date of publication or language restrictions.Data extraction and synthesisStudy selection was carried out independently by two reviewers, with abstracted data and risk of bias assessment being performed using the Cochrane tool. Data on the restorations were dichotomised as acceptable' (restorations without need of replacement or repair) or 'unacceptable' (restorations presenting failures or requiring repair or replacement) after which a number of meta-analyses were conducted.ResultsTen studies were included in qualitative synthesis, and nine contributing to the meta-analyses. Six studies used a split-mouth design and four a parallel design. Seven studies used USPHS criteria, two applied the FDI criteria and one used their own. Seven studies reported restorations were placed under rubber dam isolation with the other three using cotton roll isolation. Six studies were at low risk of bias and four unclear risk of bias. GIC and CR presented similar failure patterns (Risk Difference [RD] = -0.04 (95%CI; -0.11 to 0.03) p=0.25, I2 = 51%), irrespective of follow-up period, type of GIC used, method of isolation or criteria used for assessment. GICs exhibited significantly lower values of secondary carious lesions ([RD] = 0.06 (95%CI; 0.0 to 0.10), p=0.008, I2 = 0%).ConclusionsGICs and CRs have comparable clinical performance in Class II restorations in primary molars. GICs did show superior performance in the occurrence of secondary carious lesions, especially when RM-GIC under rubber dam isolation was used.

Reminder systems during orthodontic treatment.

Data sourcesCochrane Central Register of Controlled Trials (CENTRAL), LILACS, Scopus, Web of Science, Medline and Embase.Study selectionHuman randomised controlled trials evaluating the effectiveness of reminders in orthodontics were included. Interventions including any form of participant reminder compared to a control. There were no limitations in terms of publication year, language or status. Primary outcomes measured were periodontal parameters and rate of attendance. Six secondary outcomes were also measured.Data extraction and synthesisStudy selection and data extraction were carried out independently by two reviewers, with a third reviewer utilised to resolve disagreements. Authors were also contacted if any further clarification was required with regards to missing data. Risk of bias was assessed using the Cochrane tool. Comparable outcomes were collated and analysed using a random-effects model, with corresponding 95% confidence intervals.ResultsFourteen parallel randomised controlled trials met the inclusion criteria. Only nine contributed to the meta-analyses, as five were deemed high risk of bias. Of the trials, ten RCTs, six RCTs, five RCTs and four RCTs measured plaque scores, gingival scores, rate of appointment attendance, and the effectiveness of reminder on the development of white spot lesions (WSLs) respectively. Results were grouped into either short term (1-3 months) or long term (>3 months) outcomes. In the short term, gingival condition was healthier in the reminders group (SMD = -0.66 with 95% CI: -0.97 - 0.35) and a statistically significant difference favouring patients receiving reminders was also seen in terms of plaque control (SMD = -0.38 with 95% CI: -0.65 to -0.10). In the long term, similar outcomes were recorded, with a statistically significant SMD for plaque scores and gingival scores when reminders were used (SMD -1.51 with 95% CI: -2.72 to -0.30 and SMD -1.94 with 95% CI: -3.81 to -0.07 respectively). Development of WSLs and risk of failure/cancellation were also lower in the reminder group.ConclusionsThis systematic review highlights that there is moderate to high quality evidence showing the positive effect of reminders on oral hygiene and appointment adherence in orthodontic patients. The authors suggest further high quality RCTs with longer follow-ups would be beneficial to support the efficacy of this intervention.

Epitaxial graphene homogeneity and quantum Hall effect in millimeter-scale devices.

Quantized magnetotransport is observed in 5.6 × 5.6 mm2 epitaxial graphene devices, grown using highly constrained sublimation on the Si-face of SiC(0001) at high temperature (1900 °C). The precise quantized Hall resistance of [Formula: see text] is maintained up to record level of critical current Ixx = 0.72 mA at T = 3.1 K and 9 T in a device where Raman microscopy reveals low and homogeneous strain. Adsorption-induced molecular doping in a second device reduced the carrier concentration close to the Dirac point (n ≈ 1010 cm-2), where mobility of 18760 cm2/V is measured over an area of 10 mm2. Atomic force, confocal optical, and Raman microscopies are used to characterize the large-scale devices, and reveal improved SiC terrace topography and the structure of the graphene layer. Our results show that the structural uniformity of epitaxial graphene produced by face-to-graphite processing contributes to millimeter-scale transport homogeneity, and will prove useful for scientific and commercial applications.

Linkage of catalysis and 5' end recognition in ribonuclease RNase J.

In diverse bacterial species, the turnover and processing of many RNAs is mediated by the ribonuclease RNase J, a member of the widely occurring metallo-ß-lactamase enzyme family. We present crystal structures of Streptomyces coelicolor RNase J with bound RNA in pre- and post-cleavage states, at 2.27 Å and 2.80 Å resolution, respectively. These structures reveal snapshots of the enzyme cleaving substrate directionally and sequentially from the 5' terminus. In the pre-cleavage state, a water molecule is coordinated to a zinc ion pair in the active site but is imperfectly oriented to launch a nucleophilic attack on the phosphate backbone. A conformational switch is envisaged that enables the in-line positioning of the attacking water and may be facilitated by magnesium ions. Adjacent to the scissile bond, four bases are stacked in a tightly sandwiching pocket, and mutagenesis results indicate that this organization helps to drive processive exo-ribonucleolytic cleavage. Like its numerous homologues, S. coelicolor RNase J can also cleave some RNA internally, and the structural data suggest how the preference for exo- versus endo-cleavage mode is linked with recognition of the chemical status of the substrate's 5' end.

Couple Beads: An integrated method of natural family planning.

Various fertility indicators are used by natural family planning methods to identify the fertile and infertile phases of a woman's menstrual cycle: mucus observations, cycle-day probabilities, basal body temperature readings, and hormonal measures of LH and estrogen. Simplified NFP methods generally make use of a single fertility indicator such as cycle-day probabilities (Standard Days Method) or mucus observations (Billings Ovulation Method). The Couple Bead Method integrates the two simplest fertility indicators, cycle-day probabilities and mucus observations, expanding its applicability to all women, regardless of cycle regularity and length. In determining cycle-day probabilities, the Couple Bead Method relies on a new data set from ultrasound-derived determinants of gestational age that more directly define the day of conception and the fertile window. By using a visual-based system of inexpensive colored beads, the Couple Bead Method can be used by couples of all educational and income levels. Lay Summary: Natural family planning methods provide education in regard to the signs of a woman's body which indicate if she is possibly fertile or not. Two important signs are the day of her menstrual cycle and her observations of bleeding and cervical mucus or dryness. The Couple Bead Method teaches a couple how to observe these signs and chart them with a system of colored beads. The Couple Bead Method can be used by women with regular or irregular cycles. The bead sets are inexpensive and consist of a length of plastic cord, colored "pony beads" and safety pins.

SCO5745, a bifunctional RNase J ortholog, affects antibiotic production in Streptomyces coelicolor.

The bacterial RNases J are considered bifunctional RNases possessing both endo- and exonucleolytic activities. We have isolated an RNase J ortholog from Streptomyces coelicolor encoded by the gene sco5745. We overexpressed a decahistidine-tagged version of SCO5745 and purified the overexpressed protein by immobilized metal ion affinity chromatography. We demonstrated the presence of both 5'-to-3' exonucleolytic and endonucleolytic activities on the Bacillus subtilis thrS transcript. Exonucleoytic activity predominated with 5' monophosphorylated thrS, while endonucleolytic activity predominated with 5' triphosphorylated thrS. While sco5745 is the only RNase J allele in S. coelicolor, the gene is not essential. Its disruption resulted in delayed production of the antibiotic actinorhodin, overproduction of undecylprodigiosin, and diminished production of the calcium-dependent antibiotic, in comparison with the parental strain.