Molecular Crowding: The History and Development of a Scientific Paradigm.

It is now generally accepted that macromolecules do not act in isolation but "live" in a crowded environment, that is, an environment populated by numerous different molecules. The field of molecular crowding has its origins in the far 80s but became accepted only by the end of the 90s. In the present issue, we discuss various aspects that are influenced by crowding and need to consider its effects. This Review is meant as an introduction to the theme and an analysis of the evolution of the crowding concept through time from colloidal and polymer physics to a more biological perspective. We introduce themes that will be more thoroughly treated in other Reviews of the present issue. In our intentions, each Review may stand by itself, but the complete collection has the aspiration to provide different but complementary perspectives to propose a more holistic view of molecular crowding.

Combining full-length gene assay and SpliceAI to interpret the splicing impact of all possible SPINK1 coding variants.

BACKGROUND: Single-nucleotide variants (SNVs) within gene coding sequences can significantly impact pre-mRNA splicing, bearing profound implications for pathogenic mechanisms and precision medicine. In this study, we aim to harness the well-established full-length gene splicing assay (FLGSA) in conjunction with SpliceAI to prospectively interpret the splicing effects of all potential coding SNVs within the four-exon SPINK1 gene, a gene associated with chronic pancreatitis. RESULTS: Our study began with a retrospective analysis of 27 SPINK1 coding SNVs previously assessed using FLGSA, proceeded with a prospective analysis of 35 new FLGSA-tested SPINK1 coding SNVs, followed by data extrapolation, and ended with further validation. In total, we analyzed 67 SPINK1 coding SNVs, which account for 9.3% of the 720 possible coding SNVs. Among these 67 FLGSA-analyzed SNVs, 12 were found to impact splicing. Through detailed comparison of FLGSA results and SpliceAI predictions, we inferred that the remaining 653 untested coding SNVs in the SPINK1 gene are unlikely to significantly affect splicing. Of the 12 splice-altering events, nine produced both normally spliced and aberrantly spliced transcripts, while the remaining three only generated aberrantly spliced transcripts. These splice-impacting SNVs were found solely in exons 1 and 2, notably at the first and/or last coding nucleotides of these exons. Among the 12 splice-altering events, 11 were missense variants (2.17% of 506 potential missense variants), and one was synonymous (0.61% of 164 potential synonymous variants). Notably, adjusting the SpliceAI cut-off to 0.30 instead of the conventional 0.20 would improve specificity without reducing sensitivity. CONCLUSIONS: By integrating FLGSA with SpliceAI, we have determined that less than 2% (1.67%) of all possible coding SNVs in SPINK1 significantly influence splicing outcomes. Our findings emphasize the critical importance of conducting splicing analysis within the broader genomic sequence context of the study gene and highlight the inherent uncertainties associated with intermediate SpliceAI scores (0.20 to 0.80). This study contributes to the field by being the first to prospectively interpret all potential coding SNVs in a disease-associated gene with a high degree of accuracy, representing a meaningful attempt at shifting from retrospective to prospective variant analysis in the era of exome and genome sequencing.

First investigation of RH gene polymorphism in patients with sickle cell disease and associated blood donors in Cameroon, Central Africa.

BACKGROUND: Although genetic polymorphism of the RH blood group system is well known in sub-Saharan Africa, national/regional specificities still remain to be described precisely. For the first time in Cameroon, Central Africa, and in order to better characterize the molecular basis driving RH phenotype variability, as well as to identify the main antigens that may be potentially responsible for alloimmunization, we sought 1) to study the RH genes in a cohort of 109 patients with sickle cell disease; 2) to study the same genes in the corresponding donors whose red blood cells (RBCs) were transfused to the patients (108 donors in 98 patients); 3) to predict RH phenotype on the basis of the molecular data and compare the results with serologic testing; and 4) to identify retrospectively patients at risk for alloimmunization. MATERIALS AND METHODS: In order to generate an exhaustive dataset, the RH genes of all patient and donor samples were systematically investigated 1) by quantitative multiplex PCR of short fluorescent fragments (QMPSF) for characterization of RHD gene zygosity and potential structural variants (SVs), and 2) by Sanger sequencing for identification of single nucleotide variants (SNVs). Subsequent to molecular analysis, the genotypes and RH phenotype were deduced and predicted, respectively, from reference databases. RESULTS: In a total of 217 Cameroonian individuals, as many as 24 and up to 22 variant alleles were identified in the RHD and RHCE genes, respectively, in addition to the reference alleles. Interestingly, 65 patients with SCD (66.3%) were assumed to be exposed to one or more undesirable RH antigen(s) with varying degrees of clinical relevance. DISCUSSION: Beyond the comprehensive report of the nature and distribution of RH variant alleles in a subset of Cameroonian patients treated by transfusion therapy, this work highlights the need for an extensive review of current practice, including routine serologic typing procedures, preferably in the near future.

Mutations in Tau Protein Promote Aggregation by Favoring Extended Conformations.

Amyloid aggregation of the intrinsically disordered protein (IDP) tau is involved in several diseases, called tauopathies. Some tauopathies can be inherited due to mutations in the gene encoding tau, which might favor the formation of tau amyloid fibrils. This work aims at deciphering the mechanisms through which the disease-associated single-point mutations promote amyloid formation. We combined biochemical and biophysical characterization, notably, small-angle X-ray scattering (SAXS), to study six different FTDP-17 derived mutations. We found that the mutations promote aggregation to different degrees and can modulate tau conformational ensembles, intermolecular interactions, and liquid-liquid phase separation propensity. In particular, we found a good correlation between the aggregation lag time of the mutants and their radii of gyration. We show that mutations disfavor intramolecular protein interactions, which in turn favor extended conformations and promote amyloid aggregation. This work proposes a new connection between the structural features of tau monomers and their propensity to aggregate, providing a novel assay to evaluate the aggregation propensity of IDPs.