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We have developed a small benchtop oligonucleotide synthesizer which allows the scientist to prepare, rapidly and economically, up to 24 oligonucleotides in one batch. We have shown that this instrument can be used for peptide synthesis, as well. The instrument is based on the centrifugation method for solid–liquid separation.

Modern proteins are remarkable polymers built from a 20-amino-acid alphabet, shaped by billions of years of evolution. Yet in Earth's prebiotic era, several amino acids - particularly the canonical basic residues lysine, arginine, and histidine - were likely scarce, unlike the more readily available acidic amino acids. Moreover, protein-length polymers were inaccessible before ribosomal synthesis emerged, and peptides were probably short, statistical, and non-templated. How the earliest proteins and enzymes emerged under these constraints remains a central question in origins-of-life research. Here, we synthesize random peptide libraries that span a broad electrostatic spectrum and systematically interrogate their properties. The data indicate that a prebiotically plausible acidic alphabet stands out in its propensity for secondary structure and higher-order soluble assembly via formation of β-sheets. These assemblies arise from highly heterogeneous sequences, plausibly reflecting the statistical diversity of early Earth peptides, and differ from amyloid structures in both solubility and morphology. Our results further show that the acidic random peptides have inherent capacity to bind certain metal ions, implying their potential to contribute to prebiotic catalysis. Using a large language model for structural prediction, we further show that peptides composed of this acidic alphabet exhibit a strong propensity for compact conformations. Altogether, this study showcases that unevolved sequences of prebiotically-abundant amino acids can readily produce foldable self-assembling polymers, potentially providing a steppingstone toward the first proteins, prior to the onset of purifying selection.

Proteolysis of short Nα-protected peptide substrates bound to polyoxyethylene-polystyrene beads releases selectively free amino sites in the enzyme-accessible ``surface'' area. The substantial majority of functional sites in the ``interior'' of the polymeric support are not reached by the enzyme and remain uncleaved (protected). Subsequent synthesis with two classes of orthogonal protecting groups-- Nα-tert-butyloxycarbonyl (Boc) and Nα-9-fluorenylmethyloxycarbonyl (Fmoc)--allows generation of two structures on the same bead. The surface structure is available for receptor interactions, whereas the corresponding interior structure is used for coding. Coding structures are usually readily sequenceable peptides. This ``shaving'' methodology was illustrated by the preparation of a peptide-encoded model peptide combinatorial library containing 1.0 × 105 members at ≈ 6-fold degeneracy. From this single library, good ligands were selected for three different receptors: anti-β -endorphin antibody, streptavidin, and thrombin, and the binding structures were deduced correctly by sequencing the coding peptides present on the same beads.

To speed drug discovery, we developed an approach for identification of individual peptides with a desired biological activity from a library containing millions of peptides. The approach uses sequential orthogonal release of chemically synthesized peptides from insoluble beads, followed by testing in solution. In this system, each bead within a library of beads has one peptide sequence, but peptide molecules are attached to the bead with three types of chemical linkers, including two linkers cleavable at different pH optima. An uncleavable linker keeps some peptide attached to the bead for sequencing positives from the solution assay. Applicability of this discovery technique was documented by identifying ligands for a monoclonal antibody and for the human platelet fibrinogen receptor, glycoprotein IIb/IIIa.

Whereas modern proteins rely on a quasi-universal repertoire of 20 canonical amino acids (AAs), numerous lines of evidence suggest that ancient proteins relied on a limited alphabet of 10 early AAs, and that the 10 late AAs were products of biosynthetic pathways. However, many non-proteinogenic AAs were also prebiotically available, which begs two fundamental questions: Why do we have the current modern amino acid alphabet, and Would proteins be able to fold into globular structures as well if different amino acids comprised the genetic code? Here, we experimentally evaluated the solubility and secondary structure propensities of several prebiotically relevant amino acids in the context of synthetic combinatorial 25-mer peptide libraries. The most prebiotically abundant linear aliphatic and basic residues were incorporated along with or in place of other early amino acids to explore these alternative sequence spaces. We show that foldability was a critical factor in the selection of the canonical alphabet. Unbranched aliphatic and short-chain basic amino acids were purged from the proteinogenic alphabet despite their high prebiotic abundance because they generate polypeptides that are over-solubilized and have low packing efficiency. Surprisingly, we find that the inclusion of a short-chain basic amino acid also decreases polypeptides secondary structure potential. Our results support the view that despite lacking basic residues, the early canonical alphabet was remarkably adaptive at supporting protein folding and explain why basic residues were only incorporated at a later stage of the alphabet evolution.Competing Interest StatementThe authors have declared no competing interest.Footnotes* Inclusion of additional quality control experiments of the synthesized peptide libraries

In an attempt to prepare 7-substituted 3,4-dihydroisoquinolinone family of compounds, we observed an unexpected decarboxylation. The reaction of 4-nitrohomophthalic anhydride with a Schiff base formed on solid support leads to the formation of core structure. LC-MS and 1H NMR analysis confirmed the structure of unexpected intermediate. A library of 38,400 compounds was produced using this new synthetic approach.

Construction of synthetic combinatorial libraries is described that allows for the generation of a library of motifs rather than a library of compounds. Peptide libraries based on this strategy were synthesized and screened with model targets streptavidin and anti-β-endorphin antibody. The screens resulted in observation of expected motifs providing evidence of the effectiveness of the suggested approach.

The worldwide outbreak of the novel 2019 coronavirus disease (COVID-19) has led to recognition of a new immunopathological condition: paediatric inflammatory multisystem syndrome (PIMS-TS). The Czech Republic (CZ) suffered from one of the highest incidences of individuals who tested positive during pandemic waves. The aim of this study was to analyse epidemiological, clinical, and laboratory characteristics of all cases of paediatric inflammatory multisystem syndrome (PIMS-TS) in the Czech Republic (CZ) and their predictors of severe course. We performed a retrospective-prospective nationwide observational study based on patients hospitalised with PIMS-TS in CZ between 1 November 2020 and 31 May 2021. The anonymised data of patients were abstracted from medical record review. Using the inclusion criteria according to World Health Organization definition, 207 patients with PIMS-TS were enrolled in this study. The incidence of PIMS-TS out of all SARS-CoV-2-positive children was 0.9:1,000. The estimated delay between the occurrence of PIMS-TS and the COVID-19 pandemic wave was 3 weeks. The significant initial predictors of myocardial dysfunction included mainly cardiovascular signs (hypotension, oedema, oliguria/anuria, and prolonged capillary refill). During follow-up, most patients (98.8%) had normal cardiac function, with no residual findings. No fatal cases were reported.Conclusions: A 3-week interval in combination with incidence of COVID-19 could help increase pre-test probability of PIMS-TS during pandemic waves in the suspected cases. Although the parameters of the models do not allow one to completely divide patients into high and low risk groups, knowing the most important predictors surely could help clinical management.What is Known:• Preliminary evidence, majority from relatively small, and mostly single-centre patient cohorts, has shown some insights in the basic epidemiological and clinical data of children with a paediatric inflammatory multisystem syndrome temporally associated with SARS-CoV-2 (PIMS-TS).What is New:• To our knowledge, this is the unique published national population-wide cohort allowing to study the epidemiology (including overall incidence), time gap between viral exposure and clinical symptoms of PIMS-TS, and clinical presentations and outcomes within the individual pandemic waves of COVID-19 that were characterised by various prevailing genetic variants of SARS-CoV-2.• We estimated 3-week interval as a most probable period between SARS-CoV-2 infection and PIMS-TS based on nationwide population data using cross-correlation method.

Background With the development of molecular high-throughput assays (i.e. next generation sequencing), the knowledge on the contribution of genetic and epigenetic alterations to the etiology of inherited endocrine disorders has massively expanded. However, the rapid implementation of these new molecular tools in the diagnostic settings makes the interpretation of diagnostic data increasingly complex. Main body This joint paper of the ENDO-ERN members aims to overview chances, challenges, limitations and relevance of comprehensive genetic diagnostic testing in rare endocrine conditions in order to achieve an early molecular diagnosis. This early diagnosis of a genetically based endocrine disorder contributes to a precise management and helps the patients and their families in their self-determined planning of life. Furthermore, the identification of a causative (epi)genetic alteration allows an accurate prognosis of recurrence risks for family planning as the basis of genetic counselling. Asymptomatic carriers of pathogenic variants can be identified, and prenatal testing might be offered, where appropriate. Conclusions The decision on genetic testing in the diagnostic workup of endocrine disorders should be based on their appropriateness to reliably detect the disease-causing and –modifying mutation, their informational value, and cost-effectiveness. The future assessment of data from different omic approaches should be embedded in interdisciplinary discussions using all available clinical and molecular data.

Aim. GCK-MODY is an autosomal dominant form of diabetes caused by heterozygous mutations in the glucokinase gene leading to a lifelong mild hyperglycemia. The risk of macrovascular complications is considered low, but studies are limited. We, therefore, investigated the carotid intima-media thickness (CIMT) as an indicator of macrovascular complications in a group of patients with GCK-MODY. Methods. Twenty-seven GCK mutation carriers and 24 controls recruited among their first-degree relatives were compared, all aging over 35 years. The CIMT was tested using a high-resolution B-mode carotid ultrasonography. Medical history, anthropometry, and biochemical blood workup were obtained. Results. The mean CIMT was 0.707 ± 0.215 mm (mean ± SD) in GCK mutation carriers and 0.690 ± 0.180 mm in control individuals. When adjusted for age, gender, and family status, the estimated mean difference in CIMT between the two groups increased to 0.049 mm (P=0.19). No difference was detected for other characteristics, with the exception of fasting blood glucose (GCK-MODY 7.6 mmol/L ± 1.2 (136.4 mg/dL); controls 5.3 mmol/L ± 0.3 (95.4 mg/dL); P<0.0001) and glycated hemoglobin HbA1c (GCK-MODY 6.9% ± 1.0%, 52 mmol/mol ± 10; controls 5.7% ± 0.4%, 39 mmol/mol ± 3; P<0.0001). The frequency of myocardial infarction and ischemic stroke did not differ between groups. Conclusion. Our data indicate that the persistent hyperglycemia in GCK-MODY is associated with a low risk of developing diabetic macrovascular complications.