Feature-based molecular networking in the GNPS analysis environment.

Molecular networking has become a key method to visualize and annotate the chemical space in non-targeted mass spectrometry data. We present feature-based molecular networking (FBMN) as an analysis method in the Global Natural Products Social Molecular Networking (GNPS) infrastructure that builds on chromatographic feature detection and alignment tools. FBMN enables quantitative analysis and resolution of isomers, including from ion mobility spectrometry.

Total Synthesis and Biological Evaluation of Paenilamicins from the Honey Bee Pathogen Paenibacillus larvae.

Paenilamicins are a group of complex polycationic peptide secondary metabolites with antibacterial and antifungal activities produced by the devastating honey bee brood pathogen Paenibacillus larvae causing the lethal brood disease American Foulbrood (AFB). Here, we report the convergent total synthesis and structural revision of paenilamicin B2. Specific stereoisomers of paenilamicin B2 were synthesized for unambiguous confirmation of the natural product structure and for evaluation of biological activities. These studies revealed the N-terminal fragment of paenilamicin as an important pharmacophore. Infection assays using bee larvae and the insect pathogen Bacillus thuringiensis demonstrated that paenilamicins outcompete bacterial competitors in the ecological niche of P. larvae. Finally, we show first data that classifies paenilamicins as potential ribosome inhibitors. Hence, our synthesis route is a further step for understanding the pathogenicity of P. larvae and for thorough structure-activity-relationship as well as mode-of-action studies in the near future.

Genetic potential for changes in breeding systems: Predicted and observed trait changes during artificial selection for male and female allocation in a gynodioecious species.

PREMISE: Evolution of separate sexes from hermaphroditism often proceeds through gynodioecy, but genetic constraints on this process are poorly understood. Genetic (co-)variances and between-sex genetic correlations were used to predict evolutionary responses of multiple reproductive traits in a sexually dimorphic gynodioecious species, and predictions were compared with observed responses to artificial selection. METHODS: Schiedea (Caryophyllaceae) is an endemic Hawaiian lineage with hermaphroditic, gynodioecious, subdioecious, and dioecious species. We measured genetic parameters of Schiedea salicaria and used them to predict evolutionary responses of 18 traits in hermaphrodites and females in response to artificial selection for increased male (stamen) biomass in hermaphrodites or increased female (carpel, capsule) biomass in females. Observed responses over two generations were compared with predictions in replicate lines of treatments and controls. RESULTS: In only two generations, both stamen biomass in hermaphrodites and female biomass in females responded markedly to direct selection, supporting a key assumption of models for evolution of dioecy. Other biomass traits, pollen and ovule numbers, and inflorescence characters important in wind pollination evolved indirectly in response to selection on sex allocation. Responses generally followed predictions from multivariate selection models, with some responses unexpectedly large due to increased genetic correlations as selection proceeded. CONCLUSIONS: Results illustrate the power of artificial selection and utility of multivariate selection models incorporating sex differences. They further indicate that pollen and ovule numbers and inflorescence architecture could evolve in response to selection on biomass allocation to male versus female function, producing complex changes in plant phenotype as separate sexes evolve.

Bioactive Peptide Natural Products as Lead Structures for Medicinal Use.

The need for new drugs for the treatment of various diseases is enormous. From the previous century until the present, numerous peptide and peptide-derived natural products have been isolated from bacteria and fungi. Hence, microorganisms play a pivotal role as sources for novel drugs with an emphasis on anti-infective agents. Various disciplines from biology, chemistry, and medicine are involved in early stages of the search for peptide natural products including taxonomy, microbiology, bioanalytics, bioinformatics, and medicinal chemistry. Under biochemical aspects, small peptide drugs are basically either ribosomally synthesized and post-translationally modified (RiPPs) or synthesized by multimodular nonribosomal peptide synthetases (NRPSs). Within the context of current developments on bioactive peptide natural products, this Account predominantly highlights recent discoveries, approaches, and research from our laboratory on RiPPs and NRPSs from bacteria and fungi. In our search for peptides showing bioactivities of interest, different approaches were applied: classical screening, in silico prediction, in vitro reconstitution, site-directed mutagenesis, chemoenzymatics, heterologous expression, and total synthesis including structure-activity relationship (SAR) studies in the research on the labyrinthopeptins, albicidin, and the cyclodepsipeptides (CDPs). The ribosomally synthesized labyrinthopeptins, class III lanthipeptides, which have been discovered in a classical screening campaign, display highly attractive antiallodynic (against neuropathic pain caused by dysfunction of the nervous system) and antiviral activities. Therefore, the biosynthetic assembly was investigated by extensive enzymatic studies of the modifying enzymes, and site-directed mutagenesis was performed for the generation of analogs. By genome mining, other class III lanthipeptides have been uncovered, while synthetic access proved to be an unmet challenge for the labyrinthopeptins. In contrast, for the gyrase inhibitor albicidin, the establishment of a chemical synthesis followed by medicinal chemistry studies was the only viable option to gain access to derivatives. Albicidin, which has been discovered investigating plant host-pathogen interactions, has a strong activity against Gram-negative bacteria, for example, Escherichia coli and Pseudomonas aeruginosa, and a future synthetic derivative may become a lead structure for development of an anti-Gram-negative drug. The compound class of the cyclodepsipeptides contributes already two marketed drugs, enniatin (fusafungine) and emodepside. Cyclodepsipeptides show general antibacterial and antifungal effects, whereas specific insecticidal and anthelmintic activities provide lead structures for drug development. Hence, exploiting the chances of reprogramming NRPSs, the generation of chimeric or otherwise designed synthetases could render a new untapped structural space and thus novel bioactivities. While current developments in the fields of genomics, bioinformatics, and molecular biology facilitate the search for new natural products and the design of new peptide structures, the next decade will show which compounds have been carried on further applications and whether current developments have led to an increase in drug candidates.

Total Synthesis and Biological Assessment of Novel Albicidins Discovered by Mass Spectrometric Networking.

Natural products represent an important source of potential novel antimicrobial drug leads. Low production by microorganisms in cell culture often delays the structural elucidation or even prevents a timely discovery. Starting from the anti-Gram-negative antibacterial compound albicidin produced by Xanthomonas albilineans, we describe a bioactivity-guided approach combined with non-targeted tandem mass spectrometry and spectral (molecular) networking for the discovery of novel antimicrobial compounds. We report eight new natural albicidin derivatives, four of which bear a ß-methoxy cyanoalanine or ß-methoxy asparagine as the central α-amino acid. We present the total synthesis of these albicidins, which facilitated the unambiguous determination of the (2 S,3 R)-stereoconfiguration which is complemented by the assessment of the stereochemistry on antibacterial activity.

Mass Spectrometry-Based Visualization of Molecules Associated with Human Habitats.

The cars we drive, the homes we live in, the restaurants we visit, and the laboratories and offices we work in are all a part of the modern human habitat. Remarkably, little is known about the diversity of chemicals present in these environments and to what degree molecules from our bodies influence the built environment that surrounds us and vice versa. We therefore set out to visualize the chemical diversity of five built human habitats together with their occupants, to provide a snapshot of the various molecules to which humans are exposed on a daily basis. The molecular inventory was obtained through untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of samples from each human habitat and from the people that occupy those habitats. Mapping MS-derived data onto 3D models of the environments showed that frequently touched surfaces, such as handles (e.g., door, bicycle), resemble the molecular fingerprint of the human skin more closely than other surfaces that are less frequently in direct contact with humans (e.g., wall, bicycle frame). Approximately 50% of the MS/MS spectra detected were shared between people and the environment. Personal care products, plasticizers, cleaning supplies, food, food additives, and even medications that were found to be a part of the human habitat. The annotations indicate that significant transfer of chemicals takes place between us and our built environment. The workflows applied here will lay the foundation for future studies of molecular distributions in medical, forensic, architectural, space exploration, and environmental applications.

Paenilamicins from the honey bee pathogen Paenibacillus larvae are context-specific translocation inhibitors of protein synthesis.

The paenilamicins are a group of hybrid non-ribosomal peptide-polyketide compounds produced by the honey bee pathogen Paenibacillus larvae that display activity against Gram-positive pathogens, such as Staphylococcus aureus. While paenilamicins have been shown to inhibit protein synthesis, their mechanism of action has remained unclear. Here, we have determined structures of the paenilamicin PamB2 stalled ribosomes, revealing a unique binding site on the small 30S subunit located between the A- and P-site tRNAs. In addition to providing a precise description of interactions of PamB2 with the ribosome, the structures also rationalize the resistance mechanisms utilized by P. larvae. We could further demonstrate that PamB2 interferes with the translocation of mRNA and tRNAs through the ribosome during translation elongation, and that this inhibitory activity is influenced by the presence of modifications at position 37 of the A-site tRNA. Collectively, our study defines the paenilamicins as a new class of context-specific translocation inhibitors.

Pediatric glaucoma terminology.

Glaucoma is a term that refers to many different disorders which may be characterized by age of onset, associated ocular findings, or associated systemic malformations and diseases. Specification of the type of glaucoma, both clinically, and where applicable, molecularly aids in diagnosis, counseling, treatment, and prognosis. The objective of this report is to assist geneticists in understanding glaucoma and recognizing the importance of detailed terminology in clinical genetic diagnosis.

Complete genome sequence of Streptomyces sp. strain VNUA24, a potential antifungal bacterium isolated from soil.

Streptomyces species are important resources for secondary metabolites, including antimicrobial compounds. The Streptomyces sp. strain VNUA24 was isolated from a soil sample in Vietnam. The strain was identified as having antifungal activity against numerous fungal pathogens. Whole-genome sequencing was done to explore the biocontrol capability of this strain.

Identification and Evaluation of the Growth Promotion of Endophytic Bacteria on in vitro Potato Plants.

<b>Background and Objective:</b> Isolation and investigation of plant growth promoting bacteria on potato plants can provide significant information for the application of beneficial bacteria in potato production. This study aims to isolate and characterize endophytic bacteria isolated from potato roots. In addition, the potential application of endophytes in promoting potato growth under <i>in vitro</i> conditions was also investigated. <b>Materials and Methods:</b> The roots from 15 healthy potato plants were excised and surface sterilized by NaOCl and finally rinsed by sterilized water. The confirmed surface-sterilized roots were then aseptically cut into small fragments and spread onto the isolation media, followed by incubation at 27°C for up to 3 days. Six isolates that showed differences in colony morphology were selected for further investigation. All isolates were screened for IAA production, nitrogen fixation, and phosphate solubilization. <b>Results:</b> Five of the isolates were identified as <i>Bacillus</i> and isolate 30 was identified as <i>Paenibacillus alvei</i>. All isolates exhibited good IAA production. While Iso-27 had no nitrogen fixation activity, Iso-28 showed the highest level of nitrogen fixation activity (3.59 mg L¹), four isolates (Iso-9, Iso-10, Iso-11, Iso-28) could solubilize phosphate, ranging from 49.64 g L¹ to 67.98 mg L¹. After being inoculated with <i>in vitro</i> potato plants, isolates 9, 10, 28, 30, improved the stalk length, root number, fresh mass and dried mass of the potato plants. <b>Conclusion:</b> The four isolates can potentially be applied in <i>in vitro</i> potato culture.

Stock market volatility from the Covid-19 pandemic: New evidence from the Asia-Pacific region.

Responses from stock markets to the Covid-19 pandemic appear to change over time. Recent responses might differ as herd mentality in stock markets tends to become herd immunity to the pandemic. This study revisits the pandemic's effects on the Asia-Pacific countries' market volatility over the past 25 months. Primarily, we examine the changes of the impacts of the pandemic on volatility between the first pandemic period (2020) and the second period (January 2021-January 2022). Our findings indicate that implementing pandemic control measures helps reduce market volatility at the country and region levels. The effects of new Covid-19 cases and pandemic control measures on market volatility have been dramatically fading since 2021. The Toda-Yamamoto causality test and the panel impulse response functions from panel VAR estimation are also used for robustness analysis. Based on our findings, it appears that the current pandemic may no longer be blamed for stock market volatility in the Asia-Pacific region.

Insecticidal and Feeding Deterrent Effects of Kaempferia galanga L. and Amomum subulatum on Pieris rapae L. Larva.

<b>Background and Objective:</b> <i>Pieris rapae</i> L., is one of the most widespread and destructive pests of cruciferous plants. At present, synthetic chemical insecticide is still the main approach to control this pest despite several disadvantages to human health and the wildlife environment as well as biological resistance. To search for plants having insecticidal activity, the biological effects of two medicinal plants <i>Kaempferia galanga</i> L. and <i>Amomum subulatum </i>on <i>Pieris rapae</i> L., were investigated. <b>Materials and Methods:</b> The methanol extracts of dry rhizomes and fruits of <i>Kaempferia galanga</i> L. and <i>Amomum subulatum </i>were used to determine the mortality, feeding and oviposition deterrence of larvae and adult of <i>Pieris rapae</i> L. <b>Results:</b> <i>Kaempferia galanga</i> L. and <i>Amomum subulatum</i> exhibited insecticidal activity against <i>Pieris rapae</i> L., with LC₅₀ values of 2.11 and 11.80% (w/v), respectively. In the antifeedant test, <i>Kaempferia galanga</i> L., extract showed no significant difference with the control at the low concentration (0.5 and 1%). Whereas, with a concentration of 0.5%, <i>Amomum subulatum</i> extract demonstrated a high antifeedant effect on <i>Pieris rapae</i> L., larvae. In addition, plants treated with these two extracts reduced eggs laid by <i>Pieris rapae</i> L., in field conditions showing the oviposition deterrent properties. <b>Conclusion:</b> These results indicated that <i>Kaempferia galanga</i> L. and <i>Amomum subulatum </i>extracts have insecticidal substances against <i>Pieris rapae </i>L., which can be used for developing effective pesticides or/and oviposition deterrents for integrated pest management.

Molecular basis of antibiotic self-resistance in a bee larvae pathogen.

Paenibacillus larvae, the causative agent of the devastating honey-bee disease American Foulbrood, produces the cationic polyketide-peptide hybrid paenilamicin that displays antibacterial and antifungal activity. Its biosynthetic gene cluster contains a gene coding for the N-acetyltransferase PamZ. We show that PamZ acts as self-resistance factor in Paenibacillus larvae by deactivation of paenilamicin. Using tandem mass spectrometry, nuclear magnetic resonance spectroscopy and synthetic diastereomers, we identified the N-terminal amino group of the agmatinamic acid as the N-acetylation site. These findings highlight the pharmacophore region of paenilamicin, which we very recently identified as a ribosome inhibitor. Here, we further determined the crystal structure of PamZ:acetyl-CoA complex at 1.34 Å resolution. An unusual tandem-domain architecture provides a well-defined substrate-binding groove decorated with negatively-charged residues to specifically attract the cationic paenilamicin. Our results will help to understand the mode of action of paenilamicin and its role in pathogenicity of Paenibacillus larvae to fight American Foulbrood.

Praluzatamab Ravtansine, a CD166-Targeting Antibody-Drug Conjugate, in Patients with Advanced Solid Tumors: An Open-Label Phase I/II Trial.

PURPOSE: Praluzatamab ravtansine (CX-2009) is a conditionally activated Probody drug conjugate (PDC) comprising an anti-CD166 mAb conjugated to DM4, with a protease-cleavable linker and a peptide mask that limits target engagement in normal tissue and circulation. The tumor microenvironment is enriched for proteases capable of cleaving the linker, thereby releasing the mask, allowing for localized binding of CX-2009 to CD166. CX-2009 was evaluated in a phase I/II clinical trial for patients with advanced solid tumors. PATIENTS AND METHODS: Eligible patients had metastatic cancer receiving ≥2 prior treatments. CX-2009 was administered at escalating doses every 3 weeks (0.25-10 mg/kg) or every 2 weeks (4-6 mg/kg). Primary objective was to determine the safety profile and recommended phase II dose (RP2D). RESULTS: Of 99 patients enrolled, the most prevalent subtype was breast cancer (n = 45). Median number of prior therapies was 5 (range, 1-19). Dose-limiting toxicities were observed at 8 mg/kg every 3 weeks and 6 mg/kg every 2 weeks. On the basis of tolerability, the RP2D was 7 mg/kg every 3 weeks. Tumor regressions were observed at doses ≥4 mg/kg. In the hormone receptor-positive/HER2-nonamplified breast cancer subset (n = 22), 2 patients (9%) had confirmed partial responses, and 10 patients (45%) had stable disease. Imaging with zirconium-labeled CX-2009 confirmed uptake in tumor lesions and shielding of major organs. Activated, unmasked CX-2009 was measurable in 18 of 22 posttreatment biopsies. CONCLUSIONS: CD166 is a novel, ubiquitously expressed target. CX-2009 is the first conditionally activated antibody-drug conjugate to CD166 to demonstrate both translational and clinical activity in a variety of tumor types.

Effect of Health and Training on Ultrasensitive Cardiac Troponin in Marathon Runners.

PURPOSE: Cardiac troponin (cTn) is the gold standard biomarker for assessing cardiac damage. Previous studies have demonstrated increases in plasma cTn because of extreme exercise, including marathon running. We developed an easy-to-use, ultrasensitive assay for cardiac troponin I (cTnI) by combining single-molecule counting (SMC™) technology with dried blood spot (DBS) collection techniques and validated the assay on a cohort of marathon runners by correlating postmarathon cTnI elevations with training or risk variables. METHODS: An SMC-DBS method was developed for accurate and reproducible measurement of cTnI in fingerstick whole blood. Samples were collected from 42 runners both before and immediately after running a marathon. A similar collection was obtained from 22 non-running control individuals. Pre- and postrace questionnaires containing health and training variables were correlated with cTnI concentration. RESULTS: The assay quantified cTnI in all controls and marathon runners, both before and after the race. Prerace concentrations were significantly higher in marathon runners vs controls (median 3.1 vs 0.4 pg/mL; P < 0.0001). Immediate postmarathon concentrations were increased in 98% of runners (median elevation, 40.5 pg/mL; P < 0.001), including many above traditional cutoffs for acute myocardial infarction. Several health and training variables trended toward significant correlation with cTnI elevations. CONCLUSION: While further studies are needed to better understand the mechanisms and clinical implications of exercise-induced cTnI elevations, the present study suggests several variables that may be associated with such elevations and demonstrates a simple, cost-effective method for monitoring cTnI during exercise, managing chronic disease, and/or for assessing risk in large populations.

Expanding the Utility of High-Sensitivity Dried Blood Spot Immunoassay Testing with Single Molecule Counting.

BACKGROUND: Dried blood spot (DBS) testing has been used for years in newborn screening and for other applications when obtaining blood by venipuncture is impractical or expensive. However, several technical challenges have restricted the use of DBS testing to qualitative assays or to analytes that are present in relatively high concentrations. The application of high-sensitivity detection using single molecule counting (SMC™) technology can potentially overcome the limitations of DBS as specimen source. METHODS: A method was developed for reproducibly collecting, storing, and subsequently reconstituting DBS samples to be used with assays based on the SMC technology. Before extraction, DBS samples were scanned, and the blood spot area was calculated to normalize for sample volume and spot variability. DBS sample extraction was done using an efficient high-salt extraction buffer. DBS samples were tested using SMC-based cardiac troponin I (cTnI), prostate-specific antigen (PSA), and C-reactive protein (CRP) assays. RESULTS: The SMC-DBS assays showed reproducible sensitivity, precision, and the stability required for quantifying low-abundance biomarkers. These assays were not significantly impacted by normal variations in hematocrit or sample collection technique. Correlation coefficients obtained from method comparisons between SMC-DBS and laboratory-developed tests or Food and Drug Administration-cleared tests using traditional sample types were 1.08, 1.04, and 0.99 for cTnI, PSA, and high-sensitivity CRP, respectively. CONCLUSIONS: Combining DBS finger-stick blood collection with next-generation immunoassay technology will aid the expansion of DBS testing to protein biomarkers that are in low abundance or to low-volume samples, and will enable the development and adoption of DBS testing to far-reaching applications.

Physician Beware: Severe Cyanide Toxicity from Amygdalin Tablets Ingestion.

Despite the risk of cyanide toxicity and lack of efficacy, amygdalin is still used as alternative cancer treatment. Due to the highly lethal nature of cyanide toxicity, many patients die before getting medical care. Herein, we describe the case of a 73-year-old female with metastatic pancreatic cancer who developed cyanide toxicity from taking amygdalin. Detailed history and physical examination prompted rapid clinical recognition and treatment with hydroxocobalamin, leading to resolution of her cyanide toxicity. Rapid clinical diagnosis and treatment of cyanide toxicity can rapidly improve patients' clinical outcome and survival. Inquiries for any forms of ingestion should be attempted in patients with clinical signs and symptoms suggestive of poisoning.

Corrigendum: Development and validation of a prognostic model for predicting 30-day mortality risk in medical patients in emergency department (ED).

This corrects the article DOI: 10.1038/srep46474.

Development and validation of a prognostic model for predicting 30-day mortality risk in medical patients in emergency department (ED).

The primary aim of this prospective study is to develop and validate a new prognostic model for predicting the risk of mortality in Emergency Department (ED) patients. The study involved 1765 patients in the development cohort and 1728 in the validation cohort. The main outcome was mortality up to 30 days after admission. Potential risk factors included clinical characteristics, vital signs, and routine haematological and biochemistry tests. The Bayesian Model Averaging method within the Cox's regression model was used to identify independent risk factors for mortality. In the development cohort, the incidence of 30-day mortality was 9.8%, and the following factors were associated with a greater risk of mortality: male gender, increased respiratory rate and serum urea, decreased peripheral oxygen saturation and serum albumin, lower Glasgow Coma Score, and admission to intensive care unit. The area under the receiver operating characteristic curve for the model with the listed factors was 0.871 (95% CI, 0.844-0.898) in the development cohort and 0.783 (95% CI, 0.743-0.823) in the validation cohort. Calibration analysis found a close agreement between predicted and observed mortality risk. We conclude that the risk of mortality among ED patients could be accurately predicted by using common clinical signs and biochemical tests.