A vending machine for drug-like molecules - automated synthesis of virtual screening hits.

As a result of high false positive rates in virtual screening campaigns, prospective hits must be synthesised for validation. When done manually, this is a time consuming and laborious process. Large "on-demand" virtual libraries (>7 × 1012 members), suitable for preparation using capsule-based automated synthesis and commercial building blocks, were evaluated to determine their structural novelty. One sub-library, constructed from iSnAP capsules, aldehydes and amines, contains unique scaffolds with drug-like physicochemical properties. Virtual screening hits from this iSnAP library were prepared in an automated fashion for evaluation against Aedes aegypti and Phytophthora infestans. In comparison to manual workflows, this approach provided a 10-fold improvement in user efficiency. A streamlined method of relative stereochemical assignment was also devised to augment the rapid synthesis. User efficiency was further improved to 100-fold by downscaling and parallelising capsule-based chemistry on 96-well plates equipped with filter bases. This work demonstrates that automated synthesis consoles can enable the rapid and reliable preparation of attractive virtual screening hits from large virtual libraries.

Reagent-Based Scaffold Diversity for DNA-Encoded Library Design: Solid Phase Synthesis of DNA-Tagged sp3-Rich Heterocycles by SnAP Chemistry.

Reactions that require strictly dry conditions are challenging to translate to a DNA-encoded library format. Controlled pore glass solid support-connected DNA oligonucleotide-aldehyde conjugates could be condensed with SnAP reagents and cyclized to various sp3-rich heterocycles. The Boc-group of products provided a handle for product purification, and its facile removal under acidic conditions was tolerated by a chemically stabilized barcode. The reaction provides reagent-based scaffold diversity with functionalities for further library synthesis.

An integrated console for capsule-based, automated organic synthesis.

The current laboratory practices of organic synthesis are labor intensive, impose safety and environmental hazards, and hamper the implementation of artificial intelligence guided drug discovery. Using a combination of reagent design, hardware engineering, and a simple operating system we provide an instrument capable of executing complex organic reactions with prepacked capsules. The machine conducts coupling reactions and delivers the purified products with minimal user involvement. Two desirable reaction classes - the synthesis of saturated N-heterocycles and reductive amination - were implemented, along with multi-step sequences that provide drug-like organic molecules in a fully automated manner. We envision that this system will serve as a console for developers to provide synthetic methods as integrated, user-friendly packages for conducting organic synthesis in a safe and convenient fashion.

New methods for confocal imaging of infection threads in crop and model legumes.

BACKGROUND: The formation of infection threads in the symbiotic infection of rhizobacteria in legumes is a unique, fascinating, and poorly understood process. Infection threads are tubes of cell wall material that transport rhizobacteria from root hair cells to developing nodules in host roots. They form in a type of reverse tip-growth from an inversion of the root hair cell wall, but the mechanism driving this growth is unknown, and the composition of the thread wall remains unclear. High resolution, 3-dimensional imaging of infection threads, and cell wall component specific labelling, would greatly aid in our understanding of the nature and development of these structures. To date, such imaging has not been done, with infection threads typically imaged by GFP-tagged rhizobia within them, or histochemically in thin sections. RESULTS: We have developed new methods of imaging infection threads using novel and traditional cell wall fluorescent labels, and laser confocal scanning microscopy. We applied a new Periodic Acid Schiff (PAS) stain using rhodamine-123 to the labelling of whole cleared infected roots of Medicago truncatula; which allowed for imaging of infection threads in greater 3D detail than had previously been achieved. By the combination of the above method and a calcofluor-white counter-stain, we also succeeded in labelling infection threads and plant cell walls separately, and have potentially discovered a way in which the infection thread matrix can be visualized. CONCLUSIONS: Our methods have made the imaging and study of infection threads more effective and informative, and present exciting new opportunities for future research in the area.

Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome

Programmed ribosomal frameshifting is the key event during translation of the SARS-CoV-2 RNA genome allowing synthesis of the viral RNA-dependent RNA polymerase and downstream viral proteins. Here we present the cryo-EM structure of the mammalian ribosome in the process of translating viral RNA paused in a conformation primed for frameshifting. We observe that the viral RNA adopts a pseudoknot structure lodged at the mRNA entry channel of the ribosome to generate tension in the mRNA that leads to frameshifting. The nascent viral polyprotein that is being synthesized by the ribosome paused at the frameshifting site forms distinct interactions with the ribosomal polypeptide exit tunnel. We use biochemical experiments to validate our structural observations and to reveal mechanistic and regulatory features that influence the frameshifting efficiency. Finally, a compound previously shown to reduce frameshifting is able to inhibit SARS-CoV-2 replication in infected cells, establishing coronavirus frameshifting as target for antiviral intervention.

Super-Resolution Fluorescence Imaging of Arabidopsis thaliana Transfer Cell Wall Ingrowths using Pseudo-Schiff Labelling Adapted for the Use of Different Dyes.

To understand plant growth and development, it is often necessary to investigate the organization of plant cells and plant cell walls. Plant cell walls are often fluorescently labeled for confocal imaging with the dye propidium iodide using a pseudo-Schiff reaction. This reaction binds free amine groups on dye molecules to aldehyde groups on cellulose that result from oxidation with periodic acid. We tested a range of fluorescent dyes carrying free amine groups for their ability to act as pseudo-Schiff reagents. Using the low-pH solution historically used for the Schiff reaction, these alternative dyes failed to label cell walls of Arabidopsis cotyledon vascular tissue as strongly as propidium iodide but replacing the acidic solution with water greatly improved fluorescence labeling. Under these conditions, rhodamine-123 provided improved staining of plant cell walls compared to propidium iodide. We also developed protocols for pseudo-Schiff labeling with ATTO 647N-amine, a dye compatible for super-resolution Stimulated Emission Depletion (STED) imaging. ATTO 647N-amine was used for super-resolution imaging of cell wall ingrowths that occur in phloem parenchyma transfer cells of Arabidopsis, structures whose small size is only slightly larger than the resolution limit of conventional confocal microscopy. Application of surface-rendering software demonstrated the increase in plasma membrane surface area as a consequence of wall ingrowth deposition and suggests that STED-based approaches will be useful for more detailed morphological analysis of wall ingrowth formation. These improvements in pseudo-Schiff labeling for conventional confocal microscopy and STED imaging will be broadly applicable for high-resolution imaging of plant cell walls.

Antimicrobial Activity of the Quinoline Derivative HT61 against Staphylococcus aureus Biofilms.

Staphylococcus aureus biofilms are a significant problem in health care settings, partly due to the presence of a nondividing, antibiotic-tolerant subpopulation. Here we evaluated treatment of S. aureus UAMS-1 biofilms with HT61, a quinoline derivative shown to be effective against nondividing Staphylococcus spp. HT61 was effective at reducing biofilm viability and was associated with increased expression of cell wall stress and division proteins, confirming its potential as a treatment for S. aureus biofilm infections.

Stereoretentive Etherification of an α-Aryl-ß-amino Alcohol Using a Selective Aziridinium Ring Opening for the Synthesis of AZD7594.

A selective aziridinium ring-opening was used to etherify an α-aryl-ß-amino alcohol with stereochemical retention. This transformation was achieved in a biphasic system to address phenoxide solubility and the formation of a sulfonate ester impurity. The protecting group strategy was directed by a stability study of the activated α-aryl-ß-amino alcohol in this system. Process analytical techniques were used to establish reaction understanding, and mixing on large scale was modeled in silico. The process provided a selective and efficient method of preparing the nonsteroidal, inhaled selective glucocorticoid receptor modulator AZD7594.

Structural diversification of the aminobicyclo[4.3.0]nonane skeleton using alkynylsilyl-derived allylic trichloroacetimidates.

The amino substituted bicyclo[4.3.0]nonane is a molecular scaffold found in a wide range of natural products and medicinal agents. Despite this, synthetic methods for the general preparation of this structural motif are sparse. Here we evaluate a diastereoselective approach for the preparation of vinylsilyl derived aminobicyclo[4.3.0]nonanes using a one-pot multi-bond forming process involving a Pd(ii)-catalysed Overman rearrangement, a Ru(ii)-catalysed ring closing enyne metathesis reaction, followed by a hydrogen bonding directed Diels-Alder reaction. We show that a benzyldimethylsilyl-substituted alkene analogue is amenable to further functionalisation and the late stage generation of diverse sp3-rich, drug-like aminobicyclo[4.3.0]nonane scaffolds with up to six stereogenic centres.

Evaluación del impacto del desayuno escolar en el estado nutricional de los escolares / Assessment of school breakfast impact on the nutritional status of the schoolchildren

La carrera de Nutrición y Dietética, evaluó el impacto del desayuno escolar proporcionado por el Gobierno Municipal de La Paz, durante los periodos 2000 a 2001, para tal efecto, se desarrollaron siete componentes: evaluación del estado nutricional, consumo alimentario, análisis sensorial, aceptabilidad de la ración, promoción del desayuno escolar, educación y evaluación del impacto de la ración del desayuno escolar

Suction dressings in total knee arthroplasty--an alternative to deep suction drainage.

A new technique is described for dressing of surgical wounds in total knee arthroplasty that is a combination of a semi-permeable dressing and suction drainage. This technique has been used in 100 consecutive cases and drainage was collected in 92. The average volume was 198 ml (range 30 to 850 ml). There was no superficial or deep sepsis. Haematoma formation causing moderate soft tissue tension and some patient discomfort was noted in 9 knees. This form of postoperative wound management retains the nursing and hygiene advantages of deep suction drainage, whilst avoiding the patient discomfort and potential complication possibilities associated with deep internal drainage.

A synthetic vitreous fiber (SVF) occupational exposure database: implementing the SVF Health and Safety Partnership Program.

The Health and Safety Partnership Program is a voluntary workplace safety program for workers involved in the manufacture, fabrication, installation, and removal of glass wool and mineral wool products. This article describes one element of this Partnership Program, the development of an occupational exposure database that characterizes exposures by fiber type, industry sector, product type, and job description. Approximately 6000 exposure samples are included in the database, most of which were collected over the past decade, making it the most extensive and recent exposure data set on record for glass wool and mineral wool. The development of this database, as well as the initial results for exposure measurements segmented by product type and/or job description, are described. The current database shows that most applications and uses of glass wool and mineral wool involve exposures below the voluntary 1 f/cc permissible exposure limit, although some specific product types and job descriptions involve average exposures approaching the 1 f/cc limit.

A prospective, randomized, controlled evaluation of peripheral nerve stimulation versus standard clinical dosing of neuromuscular blocking agents in critically ill patients.

OBJECTIVES: To determine if vecuronium doses individualized by peripheral nerve stimulation are lower than those doses chosen by standard clinical techniques; and to determine whether patients monitored by peripheral nerve stimulation exhibit shorter recovery times and less prolonged neuromuscular blockade after discontinuation of vecuronium than control patients. DESIGN: A prospective, randomized, controlled, single-blind trial. SETTING: Two ten-bed medical intensive care units of a 937-bed tertiary care, not-for-profit, teaching hospital and health system. PATIENTS: Mechanically ventilated patients requiring continuous neuromuscular blockade as part of their therapy. INTERVENTIONS: After obtaining written, informed consent and baseline neurologic examinations, patients were randomized to treatment, where dosing was individualized by peripheral nerve stimulation or standard clinical assessment. Doses in the peripheral nerve stimulation group were adjusted to 90% blockade (Train-of-Four of 1/4). The standard clinical dosing group received doses individualized to clinical response by the medical team (blinded to Train-of-Four). Differences between groups were evaluated by Wilcoxon matched-pairs signed rank test. MEASUREMENTS AND MAIN RESULTS: A total of 77 patients (35 standard clinical patients vs. 42 peripheral nerve stimulation patients) were enrolled in the study. Despite no difference in initial doses and time to reach 90% blockade or clinical response between groups, the peripheral nerve stimulation group used less drug than the standard clinical group (0.040 +/- 0.028 vs. 0.070 +/- 0.030 mg/kg/hr, respectively, p = .001). The total cumulative amount of vecuronium for the episode of paralysis was greater in the control group (285.8 +/- 246.6 vs. 137.1 +/- 106.4 mg, p = .001). The peripheral nerve stimulation group recovered neuromuscular function (relative risk of 1.85, with 95% confidence interval [CI] of 1.02-3.35, p = .039) and spontaneous ventilation (relative risk of 1.86, 95% CI 1.00-3.45, p = .047) faster than the control group. In patients, adjusting for renal dysfunction, the likelihood of a faster recovery in the peripheral nerve stimulation group increased for neuromuscular function (relative risk of 1.89, 95% CI of 1.07-3.32, p = .018) and spontaneous ventilation (relative risk of 2.27, 95% CI of 1.23-4.21, p = .019). Patients with combined renal and liver failure similarly demonstrated a faster recovery in the peripheral nerve stimulation group. The recovery was affected to a lesser extent by adjusting for concurrent aminoglycoside and corticosteroid administration. CONCLUSIONS: Use of peripheral nerve stimulation for monitoring the degree of blockade and adjusting drug doses in continuously paralyzed critically ill medical patients results in lower doses of vecuronium to maintain a desired depth of paralysis, and allows a faster recovery of neuromuscular function and spontaneous ventilation.

Economic impact of prolonged motor weakness complicating neuromuscular blockade in the intensive care unit.

OBJECTIVE: We compared a case-series of ten patients who developed prolonged neuromuscular weakness after continuous, nondepolarizing, neuromuscular blockade with a group of controls without neuromuscular weakness to determine the economic impact of the neuromuscular weakness. DESIGN: Frequency-matched case control trial. SETTING: Medical and surgical intensive care units of a 937-bed tertiary care, university-affiliated teaching hospital. PATIENTS: Ten patients developed prolonged neuromuscular weakness after continuous administration of nondepolarizing neuromuscular blockers. Ten patients from a 1994 drug utilization database who did not develop motor weakness after paralysis were identified to serve as controls. MEASUREMENTS AND MAIN RESULTS: The medical and accounting records of the patients were retrospectively reviewed. Charge data were obtained from patient accounts. Institutional ratios to convert charges to full costs and marginal costs were obtained from the Hospital Finance Department of Henry Ford Hospital. The economic impact of the diagnosis and recovery of the motor weakness was estimated for the intensive care unit (ICU) and hospital stays and compared with those values for control patients. Median hospital charges (excluding rehabilitation), totaling $91,476, were attributed to the patients who developed neuromuscular weakness and included charges for neuromuscular blocking agents, continuous mechanical ventilation, ICU and hospital beds, neurologic studies, and physical therapy services. In the control patients, median charges were $22,191 (p = .001). The total median cost differential for a patient in the neuromuscular weakness group was in excess of $66,713 (95% confidence interval $23,485 to $189,214, p = .001). Significant differences were also found for patient charges, full costs, and marginal costs for mechanical ventilation (p = .002), neurologic studies (p = .014), as well as ICU (p = .002) and hospital (p = .001) stays. CONCLUSIONS: The development of motor weakness was associated with an increase in ICU and hospital stays, continued mechanical ventilation, and disproportionate healthcare expenditures in excess of $66,000 per patient. A prospective evaluation of the true prevalence of neuromuscular weakness after neuromuscular blockade and of the costs to the healthcare system is needed.

Recurrent varicella-zoster virus infections in apparently immunocompetent children.

Fourteen generally healthy children (5 females, 9 males, ages 18 months to 13 years) who have developed 2 to 5 attacks of chickenpox are described. Herpes zoster also occurred in 2 of 14 children. No case of chickenpox was severe or associated with complications. General studies of immunoglobulins, specific antibodies to immunization agents, complement and lymphocyte subpopulation number and function indicated that 1 of 14 had low serum IgA and 3 of 14 lacked antibody to 1 (n = 2) or 2 (n = 1) immunization agents. Varicella-zoster virus (VZV)-specific immune studies showed that the children developed VZV-antibody titers by enzyme-linked immunosorbent assay of 1:640 to 1:10 240. By immunoblot assay all appeared to develop a normal spectrum of antibodies to individual VZV proteins. All but one developed VZV cellular immune responses with stimulation indices ranging from 3.6 to 174. Sequential follow-up of 8 patients revealed 1 who became seronegative and 2 who lost VZV cell-mediated immune responses. Chickenpox may recur more frequently than is generally recognized. General and VZV-specific immune investigations are unlikely to indicate a reason.

Somatosensory evoked potential measurements in percutaneous fluid aspiration from intraspinal cystic lesions.

Two patients were evaluated with somatosensory evoked potentials (SSEPs) before and after CT-guided fluid aspiration from cystic intraspinal lesions. Our objective was to use the information and the clinical response to this procedure to determine the suitability of the patients for surgical intervention. In both patients, one with a pseudomeningocele and the other with a subarachnoid cyst, improvement in their symptoms corresponded to significant changes in their SSEPs. We believe this technique may be useful in selected cases when it is unclear whether cystic intraspinal fluid collections are responsible for the patient's symptoms. An increase in amplitude and/or decrease in latency of SSEPs, along with clinical improvement after fluid aspiration, may indicate the potential benefit of surgical intervention.

Necrobiosis lipoidica diabeticorum: a clinicopathologic study.

Necrobiosis lipoidica diabeticorum is an unusual dermatologic condition with a characteristic clinical appearance and a clear association with diabetes mellitus. There is currently no treatment that reverses the atrophic changes associated with this lesion. We have carried out a clinicopathologic study on 15 subjects and, in addition, have reviewed 10 further biopsy specimens of necrobiosis lipoidica diabeticorum. We found a frequent association of necrobiosis lipoidica diabeticorum with other chronic complications of diabetes mellitus, including limited joint mobility. It is possible that nonenzymatic glucosylation or other changes in collagen may be important in the etiology of necrobiosis lipoidica diabeticorum and the limited joint mobility. We confirmed that cutaneous anesthesia is usually present in the necrobiosis lipoidica diabeticorum lesions. With the use of an antibody to S100 protein and an immunohistochemical method, there was an apparent decreased number of nerves in the skin lesions. We suggest that sensory loss results from local destruction of cutaneous nerves by the inflammatory process. Finally, in six elliptical biopsies extending into clinically normal skin, we demonstrated that the inflammatory infiltrate of necrobiosis lipoidica diabeticorum extended from the lesion into apparently normal skin surrounding clinically active lesions. Thus, intradermal steroids might be administered to perilesional areas surrounding active lesions in the hope of halting progression.