Accelerating Pharmaceutical Process Development with an Acoustic Droplet Ejection-Multiple Reaction Monitoring-Mass Spectrometry Workflow.

Fast-paced pharmaceutical process developments (e.g., high-throughput experimentation, directed evolution, and machine learning) involve the introduction of fast, sensitive, and accurate analytical assays using limited sample volumes. In recent years, acoustic droplet ejection (ADE) coupled with an open port interface has been invented as a sampling technology for mass spectrometry, providing high-throughput nanoliter analytical measurements directly from the standard microplates. Herein, we introduce an ADE-multiple reaction monitoring-mass spectrometry (ADE-MRM-MS) workflow to accelerate pharmaceutical process research and development (PR&D). This systematic workflow outlines the selection of MRM transitions and optimization of assay parameters in a data-driven manner using rapid measurements (1 sample/s). The synergy between ADE sampling and MRM analysis enables analytical assays with excellent sensitivity, selectivity, and speed for PR&D reaction screenings. This workflow was utilized to develop new ADE-MRM-MS assays guiding a variety of industrial processes, including (1) screening of Ni-based catalysts for C-N cross-coupling reaction at 1 Hz and (2) high-throughput regioisomer analysis-enabled enzyme library screening for peptide ligation reaction. ADE-MRM-MS assays were demonstrated to deliver accurate results that are comparable to conventional liquid chromatography (LC) experiments while providing >100-fold throughput enhancement.

Music for pain relief of minor procedures in term neonates.

BACKGROUND: Music for neonatal pain has not been exclusively studied in term neonates in a well-designed trial compared to the standard of care. This study aims to assess the effectiveness of music intervention as an adjuvant in relieving acute pain in term newborns undergoing minor painful procedures. METHODS: This randomized, controlled, blinded trial included any term neonate undergoing heel prick. Both control and intervention groups received oral sucrose 2 min before heel prick. Intervention group was exposed to 'Bedtime Mozart' lullaby recorded music via bedside speakers. Pain was measured using Neonatal Infant Pain Scale (NIPS) at 1-min intervals. Investigators were blinded using noise-canceling headphones that played random music. RESULTS: A total of 100 neonates were enrolled. Mean gestational age was 39.2 weeks, and mean duration of the procedure was 113 s. Music group was found to have significantly lower pain scores [OR = 0.42 (0.31, 0.56), p < 0.001]. Baseline NIPS scores were similar across groups and there was no interaction effect between groups and time. When NIPS were categorized as pain and no pain, there continued to be statistically significant lower NIPS scores in the music group (p < 0.001). CONCLUSION: Recorded music, in addition to sucrose, is efficacious in reducing pain, encouraging its use in term neonates. IMPACT: Recorded music effectively reduces pain induced by minor procedures in term neonates. Clinical studies have shown that live and recorded music induces changes in vital signs and pain scores in the NICU's predominantly preterm population. Most of these studies were also conducted in the white ethnic population. Our study objectively proves reduction in pain scores by using recorded music in a randomized, controlled, blinded study of predominantly non-white, term neonates. Recorded music is effective in reducing acute pain in term neonates and can be widely used even in low-resource nurseries.

Azido-mediated intermolecular interactions of transition metal complexes.

The coordinated azido ligand has a variety of ways to establish intermolecular contacts whose nature is computationally analysed in this work on dimers of the [N3-Hg(CF3)] complex with different interactions involving only N⋯N contacts, or with an additional Hg⋯N contact. The applied tools include the molecular electrostatic map of the monomer, an energy decomposition analysis (EDA), a topological AIM analysis of the electron density and the study of NCI (non-covalent interactions) isosurfaces. The interactions between two azido ligands are found to be weakly stabilizing (by 0.2 to 2.7 kcal mol-1), topology-dependent and require dispersion forces to complement orbital and electrostatic stabilization. Those interactions are supplemented by the formation of simultaneous Hg⋯N secondary interactions by about -1 kcal mol-1, and by the ability of the monomer to simultaneously interact with several neighbours in the crystal structure.

Engineering Hydroxylase Activity, Selectivity, and Stability for a Scalable Concise Synthesis of a Key Intermediate to Belzutifan.

Biocatalytic oxidations are an emerging technology for selective C-H bond activation. While promising for a range of selective oxidations, practical use of enzymes catalyzing aerobic hydroxylation is presently limited by their substrate scope and stability under industrially relevant conditions. Here, we report the engineering and practical application of a non-heme iron and α-ketoglutarate-dependent dioxygenase for the direct stereo- and regio-selective hydroxylation of a non-native fluoroindanone en route to the oncology treatment belzutifan, replacing a five-step chemical synthesis with a direct enantioselective hydroxylation. Mechanistic studies indicated that formation of the desired product was limited by enzyme stability and product overoxidation, with these properties subsequently improved by directed evolution, yielding a biocatalyst capable of >15,000 total turnovers. Highlighting the industrial utility of this biocatalyst, the high-yielding, green, and efficient oxidation was demonstrated at kilogram scale for the synthesis of belzutifan.

Structure and Bonding of Halonium Compounds.

The geometrical parameters and the bonding in [D···X···D]+ halonium compounds, where D is a Lewis base with N as the donor atom and X is Cl, Br, or I, have been investigated through a combined structural and computational study. Cambridge Structural Database (CSD) searches have revealed linear and symmetrical [D···X···D]+ frameworks with neutral donors. By means of density functional theory (DFT), molecular electrostatic potential (MEP), and energy decomposition analyses (EDA) calculations, we have studied the effect of various halogen atoms (X) on the [D···X···D]+ framework, the effect of different nitrogen-donor groups (D) attached to an iodonium cation (X = I), and the influence of the electron density alteration on the [D···I···D]+ halonium bond by variation of the R substituents at the N-donor upon the symmetry, strength, and nature of the interaction. The physical origin of the interaction arises from a subtle interplay between electrostatic and orbital contributions (σ-hole bond). Interaction energies as high as 45 kcal/mol suggest that halonium bonds can be exploited for the development of novel halonium transfer agents, in asymmetric halofunctionalization or as building blocks in supramolecular chemistry.

Characterization of Fe3O4 Nanoparticles for Applications in Catalytic Activity in the Adsorption/Degradation of Methylene Blue and Esterification.

The aim of this study is to evaluate the applicability of the catalytic activity (CA) of the Fe3O4 magnetic system in the adsorption/degradation of methylene blue and esterification. The thermal decomposition method allowed the preparation of Fe3O4 nanoparticles. The crystallites of the Fe3O4 structural phase present an acicular form confirmed by X-ray diffraction. Transmission electron microscopy results identified the acicular shape and agglomeration of the nanoparticles. Mössbauer spectroscopy showed that the spectrum is composed of five components at room temperature, a hyperfine magnetic field distribution (HMFD), two sextets, a doublet, and a singlet. The presence of the HMFD means that a particle size distribution is present. Fluorescence spectroscopy studied the CA of the nanoparticles with methylene blue and found adsorption/degradation properties of the dye. The catalytic activity of the nanoparticles was evaluated in the esterification reaction by comparing the results in the presence and absence of catalyst for the reaction with isobutanol and octanol, where it is observed that the selectivity for the products MIBP and MNOP is favored in the first three hours of reaction.

Delocalized Bonding in Li2X2 Rings: Probing the Limits of the Covalent and Ionic Bonding Models.

In spite of the highly ionic character of most lithium-element bonds, the bonding within Li2X2 rings presents similarities with that found in analogous transition metal systems. They obey simple framework electron counting rules that allow us to predict whether they will form a regular ring or a squeezed one with short Li-Li or X-X distances. A combined computational and structural database analysis discloses the orbital conditions that determine the framework electron counting rules. These systems probe the borderline between the covalent and ionic bonding models since, paradoxically, a non-negligible covalent contribution of the two Li atoms (formally Li+ ions in the ionic model) to the Li-X framework bonding favors them approaching each other within bonding distance.

The resurgence of syphilis among pregnant women in southern Chile.

BACKGROUND: National rates of HIV, gonorrhoeae and syphilis have increased in Chile in recent years, but it is not known if syphilis among pregnant women (PW) is also escalating. AIM: To explore syphilis rates among PW in a southern Chilean region and to describe clinical features of mothers and offspring. PATIENTS AND METHODS: We selected PW with positive VDRL or T. pallidum micro-he-magglutination (MHA-TP) reactive tests, referred to a high-risk obstetrical pathology center between 2011 and 2019. Clinical information of mothers and their offspring was obtained from their medical records. RESULTS: Syphilis rates among PW increased from 0.4 to 7.2 per 1,000 live births in the reference center from 2013 to 2019, along with a national and regional increase in both sexes. Twenty-nine cases of PW with syphilis were identified with a median age of 28 years (interquartile range 23-32). Seventeen percent had a history of drug abuse, and 14% had previous sexually transmitted diseases. In seven cases (24%), the diagnosis occurred at delivery. Three percent of patients had a primary syphilis, 14% a secondary syphilis, 24% an early latent syphilis, and 59% a latent syphilis. All cases except one case were treated with benzathine penicillin G and the remaining with erythromycin due to allergy suspicion. Penicillin desensitization was required in two cases (7%). Treatment timing was inadequate in 10 cases (34.5%). Of 19 patients with available serological follow-up, 10 (53%) showed a significant decrease in VDRL titers (≥ 2 dilutions) and two cases had re-infection. Two patients (7%) had an abortion, two had congenital syphilis (7%), and six had premature births (21%), totaling 35% of adverse-pregnancy outcomes. CONCLUSIONS: Resurgence of syphilis among pregnant women is happening in southern Chile and is associated to adverse-pregnancy outcomes.

Onychomycosis caused by the environmental mold Neoscytalidium dimidiatum in Colombia, and in vitro antifungal susceptibility evaluation.

Neoscytalidium dimidiatum is a plant pathogen, but can also cause onychomycosis. We compared clinical and epidemiological data of cases of onychomycosis caused by N. dimidiatum and Trichophyton rubrum. We also evaluated the in vitro antifungal susceptibility of N. dimidiatum clinical isolates. It was not possible to establish any statistical differences between groups, except the place of residence and the number of affected nails. The results suggest that onychomycosis caused by N. dimidiatum is clinically similar to that caused by T. rubrum; besides, N. dimidiatum has been shown to have low sensitivity to itraconazole, but high to terbinafine. LAY SUMMARY: Cases of onychomycosis caused by Neoscytalidium dimidiatum were studied and compared to cases of onychomycosis caused by T. rubrum. The individuals affected were adults, and the clinical characteristics were not different between groups; accordingly, mycological diagnosis is mandatory.

A software to prevent delirium in hospitalised older adults: development and feasibility assessment.

BACKGROUND: non-pharmacological interventions to prevent delirium are useful in hospitalised older adults. However, they are poorly implemented in clinical practice. We aimed to develop a software for bedside use by hospitalised older adults and to improve their access to these interventions. METHODS: a transdisciplinary team composed of healthcare professionals, designers, engineers and older adults participated in the development of the software. Scrum methodology was used to coordinate the work of the team, and the software was evaluated in a feasibility study. RESULTS: a software for touchscreen mobile devices that supports Android 5.0 or later was produced, including modules for time-spatial re-orientation, cognitive stimulation, early mobilisation, sensorial support use promotion, sleep hygiene and pain management optimisation. Horizontal disposition, use of colour contrast and large interaction areas were used to improve accessibility. The software's usability and accessibility were evaluated in 34 older adults (average age 73.2 ± 9.1 years) showing that 91.1% of them got access to all the software functions without previous instructions. The clinical feasibility assessment showed that 83.3% of the 30 enrolled hospitalised patients (76 ± 8 years) completed the 5-day protocol of software usage during hospitalisation. Software use was associated with a decreased trend in delirium incidence of 5 of 32 (15.6%) at baseline to 2 of 30 (6.6%) after its implementation. CONCLUSION: a highly accessible and implementable software, designed to improve access to non-pharmacological interventions to prevent delirium in hospitalised older adults, was developed. The effectiveness of the software will be evaluated in a randomised clinical trial.

Mutations in CDKN2A and the FGFR3 genes on bladder cancer diagnosis: a systematic review and meta-analysis.

PURPOSE: To determine the association between mutations in CDKN2A and FGFR3 genes and the diagnosis of bladder carcinoma (BCa). METHODS: A systematic search strategy was carried out through MEDLINE, EMBASE, LILACS, CENTRAL and unpublished literature. We included RCTs, cohort, case-control and cross-sectional studies that involved patients > 18-year-old assessing the association between CDKN2A and FGFR3 mutated genes and BCa. The primary outcome was bladder cancer defined by histology of the sample. We assessed the risk of bias with QUADAS2 and performed a meta-analysis with Review Manager v5.3. RESULTS: We found 97 records with the search strategies. After duplicates were removed, six studies were included in meta-analysis. Regarding the association between mutated FGFR3 and bladder cancer, we found an OR 31 95% CI (15-64). However, there was no association for CDKN2A and BCa. CONCLUSION: There is a strong association between FGFR3 mutated gene and the diagnosis of bladder cancer, which has not been observed with CDKN2A. Such a result supports FGFR3 mutated gene as a promissory bladder cancer screening and monitoring biomarker.

Effectiveness of the Remeex system™ in Colombian women with recurrent stress urinary incontinence or intrinsic sphincter deficiency.

INTRODUCTION AND HYPOTHESIS: We report our experience with the Remeex system™ in women with recurrent stress urinary incontinence (SUI) or intrinsic sphincter deficiency (ISD). METHODS: A multicenter retrospective study was conducted in women who underwent an adjustable sling procedure between 2011 and 2016. We used urodynamic studies (UDS) preoperatively and the International Consultation on Incontinence Questionnaire-Short Form (ICIQ-SF) and cough stress test (CST) pre- and postoperatively. Primary outcomes were subjective (no leakage reported by the patient) and objective (no leakage during CST) cure and improvement rates (reduction of ≥4 points in ICIQ-SF). Descriptive and inferential statistics were employed. RESULTS: A total of 50 patients were included. Mean age was 62 years (SD ± 11.35). Median follow-up was 19.5 months [interquartile range (IQR) 12.95-41.38]. Urinary incontinence (UI) was described as moderate and severe by 8 (16%) and 42 (84%) patients, respectively, and 25 (50%) had stress-predominant mixed urinary incontinence (MUI). Objective and subjective cure rates were 90% and 48%, respectively, while 82% of patients achieved improvement. Impact of UI on quality of life (QoL) improved from 10 (IQR 9-10) to 2 (IQR 0-5) (p < 0.0001). Clavien-Dindo II complications occurred in 14 (28%) patients, and one (2%) had IIIa. Tape erosion occurred in one (2%) patient, and five (10%) required readjustments. Logistic regression identified MUI [odds ratio (OR) 3.3, 95% confidence interval (CI) 1.02-10.89] and vaginal atrophy (OR 4.2, 95% CI 1.06-16.03) as predictors of low subjective cure rate. CONCLUSIONS: Adjustable slings represent a valuable and safe option in the management of recurrent SUI or ISD, with improvement in QoL. Results should be carefully interpreted due to our small sample and retrospective design.

Maternal Inflammation Disrupts Fetal Neurodevelopment via Increased Placental Output of Serotonin to the Fetal Brain.

UNLABELLED: Maternal inflammation during pregnancy affects placental function and is associated with increased risk of neurodevelopmental disorders in the offspring. The molecular mechanisms linking placental dysfunction to abnormal fetal neurodevelopment remain unclear. During typical development, serotonin (5-HT) synthesized in the placenta from maternal l-tryptophan (TRP) reaches the fetal brain. There, 5-HT modulates critical neurodevelopmental processes. We investigated the effects of maternal inflammation triggered in midpregnancy in mice by the immunostimulant polyriboinosinic-polyribocytidylic acid [poly(I:C)] on TRP metabolism in the placenta and its impact on fetal neurodevelopment. We show that a moderate maternal immune challenge upregulates placental TRP conversion rapidly to 5-HT through successively transient increases in substrate availability and TRP hydroxylase (TPH) enzymatic activity, leading to accumulation of exogenous 5-HT and blunting of endogenous 5-HT axonal outgrowth specifically within the fetal forebrain. The pharmacological inhibition of TPH activity blocked these effects. These results establish altered placental TRP conversion to 5-HT as a new mechanism by which maternal inflammation disrupts 5-HT-dependent neurogenic processes during fetal neurodevelopment. SIGNIFICANCE STATEMENT: The mechanisms linking maternal inflammation during pregnancy with increased risk of neurodevelopmental disorders in the offspring are poorly understood. In this study, we show that maternal inflammation in midpregnancy results in an upregulation of tryptophan conversion to serotonin (5-HT) within the placenta. Remarkably, this leads to exposure of the fetal forebrain to increased concentrations of this biogenic amine and to specific alterations of crucially important 5-HT-dependent neurogenic processes. More specifically, we found altered serotonergic axon growth resulting from increased 5-HT in the fetal forebrain. The data provide a new understanding of placental function playing a key role in fetal brain development and how this process is altered by adverse prenatal events such as maternal inflammation. The results uncover important future directions for understanding the early developmental origins of mental disorders.

Using game authoring platforms to develop screen-based simulated functional assessments in persons with executive dysfunction following traumatic brain injury.

The assessment of functional status is a critical component of clinical neuropsychological evaluations used for both diagnostic and therapeutic purposes in patients with cognitive brain disorders. There are, however, no widely adopted neuropsychological tests that are both ecologically valid and easily administered in daily clinical practice. This discrepancy is a roadblock to the widespread adoption of functional assessments. In this paper, we propose a novel approach using a serious game authoring platform (eAdventure) for creating screen-based simulated functional assessments. We created a naturalistic functional task that consisted of preparing a cup of tea (SBS-COT) and applied the assessment in a convenience sample of eight dyads of therapists/patients with mild executive dysfunction after traumatic brain injury. We had three main aims. First, we performed a comprehensive review of executive function assessment in activities of daily living. Second, we were interested in measuring the feasibility of this technology with respect to staffing, economic and technical requirements. Third, a serious game was administered to patients to study the feasibility of this technology in the clinical context (pre-screening test). In addition, quantitative (Technology Acceptance Model (TAM) questionnaires) and qualitative (semistructured interviews) evaluations were applied to obtain user input. Our results suggest that the staffing, economic and technical requirements of the SBS-COT are feasible. The outcomes of the pre-screening test provide evidence that this technology is useful in the functional assessment of patients with executive dysfunction. In relation to subjective data, the TAM questionnaire showed good user acceptability from a professional perspective. Interview analyses with professionals and patients showed positive experiences related to the use of the SBS-COT. Our work indicates that the use of these types of authoring platforms could have positive long-term implications for neuropsychological research, opening the door to more reproducible, cooperative and efficient research by allowing the facilitated production, reuse and sharing of neuropsychological assessment tools.

SPI-9 of Salmonella enterica serovar Typhi is constituted by an operon positively regulated by RpoS and contributes to adherence to epithelial cells in culture.

The genomic island 9 (SPI-9) from Salmonella enterica serovar Typhi (S. Typhi) carries three ORFs (STY2876, STY2877, STY2878) presenting 98 % identity with a type 1 secretory apparatus (T1SS), and a single ORF (STY2875) similar to a large RTX-like protein exhibiting repeated Ig domains. BapA, the Salmonella enterica serovar Enteritidis orthologous to S. Typhi STY2875, has been associated with biofilm formation, and is described as a virulence factor in mice. Preliminary in silico analyses revealed that S. Typhi STY2875 ORF has a 600 bp deletion compared with S. Enteritidis bapA, suggesting that S. Typhi STY2875 might be non-functional. At present, SPI-9 has not been studied in S. Typhi. We found that the genes constituting SPI-9 are arranged in an operon whose promoter was up-regulated in high osmolarity and low pH in a RpoS-dependent manner. All the proteins encoded by S. Typhi SPI-9 were located at the membrane fraction, consistent with their putative role as T1SS. Furthermore, SPI-9 contributed to adherence of S. Typhi to epithelial cells when bacteria were grown under high osmolarity or low pH. Under the test conditions, S. Typhi SPI-9 did not participate in biofilm formation. SPI-9 is functional in S. Typhi and encodes an adhesin induced under conditions normally found in the intestine, such as high osmolarity. Hence, this is an example of a locus that might be designated a pseudogene by computational approaches but not by direct biological assays.

Evolution of lanthipeptide synthetases.

Lanthionine-containing peptides (lanthipeptides) are a family of ribosomally synthesized and posttranslationally modified peptides containing (methyl)lanthionine residues. Here we present a phylogenomic study of the four currently known classes of lanthipeptide synthetases (LanB and LanC for class I, LanM for class II, LanKC for class III, and LanL for class IV). Although they possess very similar cyclase domains, class II-IV synthetases have evolved independently, and LanB and LanC enzymes appear to not always have coevolved. LanM enzymes from various phyla that have three cysteines ligated to a zinc ion (as opposed to the more common Cys-Cys-His ligand set) cluster together. Most importantly, the phylogenomic data suggest that for some scaffolds, the ring topology of the final lanthipeptides may be determined in part by the sequence of the precursor peptides and not just by the biosynthetic enzymes. This notion was supported by studies with two chimeric peptides, suggesting that the nisin and prochlorosin biosynthetic enzymes can produce the correct ring topologies of epilancin 15X and lacticin 481, respectively. These results highlight the potential of lanthipeptide synthetases for bioengineering and combinatorial biosynthesis. Our study also demonstrates unexplored areas of sequence space that may be fruitful for genome mining.

Use of a phosphonate methyltransferase in the identification of the fosfazinomycin biosynthetic gene cluster.

Natural product discovery has been boosted by genome mining approaches, but compound purification is often still challenging. We report an enzymatic strategy for "stable isotope labeling of phosphonates in extract" (SILPE) that facilitates their purification. We used the phosphonate methyltransferase DhpI involved in dehydrophos biosynthesis to methylate a variety of phosphonate natural products in crude spent medium with a mixture of labeled and unlabeled S-adenosyl methionine. Mass-guided fractionation then allowed straightforward purification. We illustrate its utility by purifying a phosphonate that led to the identification of the fosfazinomycin biosynthetic gene cluster. This unusual natural product contains a hydrazide linker between a carboxylic acid and a phosphonic acid. Bioinformatic analysis of the gene cluster provides insights into how such a structure might be assembled.

Atmospheric cold plasma as a novel approach to remediating microplastics pollution in water.

Microplastics (MPs) have become an environmental and health threat to aquatic species and humans because they are small and can easily reach water bodies for municipal and agricultural uses. MPs have been traced in food commodities and products derived from animals and even found in bottles of drinking water. Current treatment techniques for permanently destroying MPs require high energy inputs and thus are generally cost-inefficient. Atmospheric cold plasma (ACP) is a low-cost energy-efficient technology to produce highly reactive species that can induce physicochemical changes in plastic polymers. This study, for the first time, used ACP as a novel method for MPs treatment. Polypropylene (PP) and low-density polyethylene (LDPE) were used to prepare model MPs. The effects of plasma working gas (oxygen, nitrogen, or their mixture) and post-ACP treatment storage (24 h) on MPs were studied. ACP treatments for 30 min successfully degraded both MPs, by 1.4-11.3% in weight. PP MPs had larger weight reduction than LDPE and the ACP of mixture gas was most effective. PP MPs also showed increased carbonyl index after treatments, to up to 6.89, indicating hydrolytic degradation. For LDPE MPs, oxygen ACP caused more oxidation, but storage did not have an enhancing effect. The results of physicochemical analyses indicated that MPs degradation by ACP was possibly mainly through oxidative and hydrolytic reactions, but further characterizations are needed. This study proves that ACP is a promising strategy to remediate MPs pollution, and thus has great potential for addressing the severe challenges of MPs that the food and agriculture sectors are currently facing.

Unveiling the Potential of Haloalkenes as Electron Density Acceptors.

We report herein, by means of structural and computational analyses, a comprehensive study of the capability of differently substituted haloalkenes to behave as electron density acceptors in noncovalent interactions. The nature of these interactions between haloalkenes and Lewis bases highly depends on the number and nature of the halogen atoms bound to the carbon-carbon double bond. When hydrogen bonds, which generally dominate for mono- and dihaloalkenes, cannot be formed, we observe the establishment of attractive interactions in which an sp2 carbon atom, belonging to an acyclic C=C double bond, plays the role of the Lewis acid via its π* antibonding orbital.