Formaldehyde (FA) is the simplest aldehyde present both in the environment and in living organisms. FA is an extremely reactive compound capable of protein crosslinking and DNA damage. For a long time, FA was considered a “biochemical waste” and a by-product of normal cellular metabolism, but in recent decades the picture has changed. As a result, the need arose for novel instruments and approaches to monitor and measure not only environmental FA in water, cosmetics, and household products, but also in food, beverages and biological samples including cells and even organisms. Despite numerous protocols being developed for in vitro and in cellulo FA assessment, many of them have remained at the “proof-of-concept” stage. We analyze the suitability of different methods developed for non-biological objects, and present an overview of the recently developed approaches, including chemically-synthesized probes and genetically encoded FA-sensors for in cellulo and in vivo FA monitoring. We also discuss the prospects of classical methods such as chromatography and spectrophotometry, and how they have been adapted in response to the demand for precise, selective and highly sensitive evaluation of FA concentration fluctuations in biological samples. The main objectives of this review is to summarize data on the main approaches for FA content measurement in liquid biological samples, pointing out the advantages and disadvantages of each method; to report the progress in development of novel molecules suitable for application in living systems; and, finally, to discuss genetically encoded FA-sensors based on existing natural biological FA-responsive elements.

Plants are a promising platform for recombinant protein production. Here we propose a novel approach to increase the level of viral vector-mediated recombinant protein synthesis. This approach is based on the hypothesis that antiviral protection is weakened during the antibacterial cellular response. We suggested that introduced to the cell foreign nuclear localized proteins, including effectors such as bacterial nucleomodulins, can interfere with the import of cellular nuclear proteins and launch antibacterial defense reactions, creating favorable conditions for cytoplasmic virus reproduction. Here, we performed synthesis of an artificial nuclear protein—red fluorescent protein (mRFP) fused with a nuclear localization sequence (NLS)—in plant cells as a mimetic of a bacterial effector. Superproduction of mRFP:NLS induced Nicotiana benthamiana γ-thionin (NbγThio) mRNA accumulation. Both NLS-containing protein synthesis and increased NbγThio expression stimulated reproduction of the viral vector based on the genome of crucifer-infecting tobacco mosaic virus (crTMV) in N. benthamiana leaves. We isolated the NbγThio gene promoter (Pr^γThio) and showed that Pr^γThio activity sharply increased in response to massive synthesis of GFP fused with NLS. We conclude that NLS-induced Pr^γThio activation and increased accumulation of Nbγthio mRNA led to the stimulation of GFP expression from crTMV: GFP vector in the transient expression system.

P62/SQSTM1 is a multifunctional scaffold protein central to selective autophagy and, more recently, recognized as a regulator of ubiquitin–proteasome system-mediated degradation of intracellular proteins. Within phase-separated condensates, p62 has been shown to recruit and sequester the proteasome, yet the molecular basis for this interaction has remained largely unknown. Our previous study demonstrated that the ‘PB1’ domain (residues 1–123) of p62 is necessary for proteasome binding. However, this long stretch is also responsible for other functions of p62, such as condensate assembly and signal transduction. Thus, it was important to define more precisely the region responsible for interaction with the proteasome. In this study, we used systematic deletion variants of p62 and biochemical assays to delineate the minimal sequence within the PB1 domain responsible for proteasome binding. Our analyses revealed a small stretch of six amino acids (residues 84–89) that bind the proteasome and are distinct from the region responsible for condensate formation. Such a precise variant can serve as a useful tool to dissect how p62–proteasome interaction affects selective degradation and probably stress response, separating it from other p62 functions. Overall, this work advances our understanding of the structural determinants underlying p62’s dual role in autophagy and UPS regulation.

Pathogenic and symbiotic bacteria secrete protein factors—nucleomodulins—to affect the host cell nucleus. During evolution, plants have acquired a great variety of defense mechanisms, including the synthesis of such antimicrobial peptides (AMPs) as defensins. We have demonstrated that the transient production of a foreign protein containing nuclear localization signal (NLS) in Nicotiana benthamiana leaves leads to the increased expression of the γ-thionin (NbγThio) that belongs to the defensin group of AMPs. We hypothesized that NbγThio is induced by the nucleomodulins of pathogenic bacteria and, in particular, in response to their NLSs. We used artificial nuclear proteins based on green fluorescent protein (GFP) fused with the human prothymosin α NLS or VirE3 NLS from Agrobacterium tumefaciens as mimetics of bacterial effectors. We demonstrated that the super-production of these NLS-containing reporters in the transient expression system in N. benthamiana leaves resulted in the increase in the NbγThio mRNA level. We isolated the NbγThio gene promoter (Pr^γThio) and created an expression vector (Pr^γThio-GUS) directing GUS synthesis in agroinfiltrated leaves. The co-expression of Pr^γThio-GUS with 35S-GFP:NLS variants led to the significant stimulation of GUS synthesis. We concluded that NbγThio gene expression is activated in response to bacterial nucleus-targeted proteins in the cell and is regulated both at the level of transcription and post-transcription stages.

QR2 (NQO2) is an intracellular flavoenzyme without a signal peptide, with expression dominated by erythrocytes and granulocytes and a reported plasma concentration of ~4.6 ng/mL. It is therefore a challenging blood analyte: it is not secreted, is predominantly cell-associated, and occurs near or below the working range of many untargeted methods. No commercially matched sandwich-antibody pair is available, and Olink does not currently include an NQO2 assay. This protocol describes a quantitative sandwich ELISA for human QR2 in serum and plasma. The assay achieves a LOQ of 2.94 ng/mL in neat serum (0.0735 ng/well), below the reported circulating concentration. Two assay features were introduced to address matrix and interference effects observed during development. Matrix-matched calibration. Undiluted serum and plasma suppress signal by approximately 6.4-fold and 8.2-fold, respectively. Dilution studies established minimum working dilutions of 1:4 for serum and 1:8 for plasma. At these dilutions, matrix effects still reduce signal by ~60%, causing buffer-only calibration to overestimate sample concentrations. Calibration is therefore performed in the same pooled matrix as the samples, using the same calibrator series in parallel buffer and matrix curves. The slope ratio provides a direct estimate of the matrix effect. Capture-free interference controls. A defined set of wells receives coating buffer without capture antibody. These wells detect signal generated independently of antigen capture. In clinical sample testing, this control identified a high-reading donor as a heterophile-antibody artefact: 85% of the donor's excess signal persisted in wells lacking the detection antibody. The calibration range uses two interleaved two-fold dilution series, with 8 of 12 points at or below 0.66 ng/well, providing increased resolution in the low-concentration range. Four-parameter logistic fits give R² > 0.999 for both buffer and matrix curves, with Hill slopes differing by <2%, consistent with a predominantly scalar matrix effect.