Establishment Genes Present on pLS20 Family of Conjugative Plasmids Are Regulated in Two Different Ways

During conjugation, a conjugative DNA element is transferred from a donor to a recipient cell via a connecting channel. Conjugation has clinical relevance because it is the major route for spreading antibiotic resistance and virulence genes. The conjugation process can be divided into different steps. The initial steps carried out in the donor cell culminate in the transfer of a single DNA strand (ssDNA) of the conjugative element into the recipient cell. However, stable settlement of the conjugative element in the new host requires at least two additional events: conversion of the transferred ssDNA into double-stranded DNA and inhibition of the hosts’ defence mechanisms to prevent degradation of the transferred DNA. The genes involved in this late step are historically referred to as establishment genes. The defence mechanisms of the host must be inactivated rapidly and—importantly—transiently, because prolonged inactivation would make the cell vulnerable to the attack of other foreign DNA, such as those of phages. Therefore, expression of the establishment genes in the recipient cell has to be rapid but transient. Here, we studied regulation of the establishment genes present on the four clades of the pLS20 family of conjugative plasmids harboured by different Bacillus species. Evidence is presented that two fundamentally different mechanisms regulate the establishment genes present on these plasmids. Identification of the regulatory sequences were critical in revealing the establishment regulons. Remarkably, whereas the conjugation genes involved in the early steps of the conjugation process are conserved and are located in a single large operon, the establishment genes are highly variable and organised in multiple operons. We propose that the mosaical distribution of establishment genes in multiple operons is directly related to the variability of defence genes encoded by the host bacterial chromosomes.

Conjugative transfer of naturally occurring plasmids in Mycolicibacterium sp.

Conjugation is considered the main horizontal gene transfer (HGT) mechanism in bacterial adaptation and evolution. In the Mycobacteriaceae family, Mycolicibacterium smegmatis has been used as the model organism for the conjugative transfer of hybrid plasmids. However, the natural conjugation process in any bacteria would involve the transfer of naturally occurring plasmids. Currently, there is a gap in this regard in relation to this abundant environmental genus of Mycobacteriaceae. Here, we performed conjugation experiments between wild Mycolicibacterium sp. strains involving naturally occurring plasmids (sizes of 21 and 274 kb), and interestingly, evidence of conjugative transfer was obtained. Thus, it is likely that conjugation occurs in Mycolicibacterium in the natural environment, representing a source of diversification and evolution in this genus of bacteria.

pLS20 is the archetype of a new family of conjugative plasmids harboured by Bacillus species

Conjugation plays important roles in genome plasticity, adaptation and evolution but is also the major horizontal gene-transfer route responsible for spreading toxin, virulence and antibiotic resistance genes. A better understanding of the conjugation process is required for developing drugs and strategies to impede the conjugation-mediated spread of these genes. So far, only a limited number of conjugative elements have been studied. For most of them, it is not known whether they represent a group of conjugative elements, nor about their distribution patterns. Here we show that pLS20 from the Gram-positive bacterium Bacillus subtilis is the prototype conjugative plasmid of a family of at least 35 members that can be divided into four clades, and which are harboured by different Bacillus species found in different global locations and environmental niches. Analyses of their phylogenetic relationship and their conjugation operons have expanded our understanding of a family of conjugative plasmids of Gram-positive origin.

How dangerous Gilbert's syndrome is

Gilbert's syndrome is a benign (functional) hyperbilirubinemia, which is based on a hereditary disorder of bilirubin metabolism, as a result of which the concentration of unbound bilirubin can increase several times. Bilirubin, being a breakdown product of hemoglobin, circulates through the bloodstream, combining with albumin molecules. Such bilirubin is called indirect. In the endoplasmic reticulum, it is conjugated; the enzyme glucuronyltransferase is responsible for this process. In Gilbert's syndrome, as a result of insufficient production of this enzyme, the conjugation process is disrupted, and, as a result, the concentration of unconjugated bilirubin increases. According to statistics, this pathological condition is observed in about 5 % of Russians. This syndrome was first described in 1901 by the French physician Augustin Nicolas Gilbert, and was subsequently named after him. The literature also contains references to this syndrome, described as «constitutional hepatic dysfunction», «familial non-hemolytic hyperbilirubinemia», «idiopathic non-conjugated hyperbilirubinemia». Gilbert's syndrome is inherited in an autosomal recessive manner; men get ill 3–4 times more often than women. A number of scientists associate this with a possible inhibitory effect of testosterone on the enzyme UDP-GT1, which breaks down bilirubin. Clinically, Gilbert's syndrome is manifested by episodes of jaundice caused by an increase in the level of unconjugated bilirubin in the blood serum. Against the background of icterus of the sclera and skin, there is increased fatigue, the appearance of a feeling of bitterness in the mouth, loss of appetite, nausea, and sometimes vomiting. The association of Gilbert's syndrome with functional disorders of the biliary tract, in particular, with gallbladder dyskinesia, is often noted.

Bioconjugated nanometals and cancer therapy: a pharmaceutical perspective

Conventional cancer chemotherapies are associated with challenges like delivering sub-therapeutic doses to target cells, lack of bioavailability, drug insolubility and nonspecific toxicity to healthy cells. Nanomedicine, an advanced process, can contribute to the development of personalized medicine for diagnosis, therapy and monitoring of cancer. The nanometals enhance drug dissolution and adhesion to targeted tumor surfaces, resulting in rapid onset of effective therapeutic action. Moreover, nanometals can be conjugated with ligands or polymers through a conjugation process, which further leads to enhanced efficiency, target specificity, improved pharmacokinetics and pharmacodynamics of the drug. This review focuses on the applications of conjugated nanometals in cancer therapy, with a special focus on noble and magnetic nanometals.

Diagnostics and Therapeutics in Targeting HER2 Breast Cancer: A Novel Approach

Breast cancer is one of the most commonly occurring cancers in women globally and is the primary cause of cancer mortality in females. BC is highly heterogeneous with various phenotypic expressions. The overexpression of HER2 is responsible for 15–30% of all invasive BC and is strongly associated with malignant behaviours, poor prognosis and decline in overall survival. Molecular imaging offers advantages over conventional imaging modalities, as it provides more sensitive and specific detection of tumours, as these techniques measure the biological and physiological processes at the cellular level to visualise the disease. Early detection and diagnosis of BC is crucial to improving clinical outcomes and prognosis. While HER2-specific antibodies and nanobodies may improve the sensitivity and specificity of molecular imaging, the radioisotope conjugation process may interfere with and may compromise their binding functionalities. Aptamers are single-stranded oligonucleotides capable of targeting biomarkers with remarkable binding specificity and affinity. Aptamers can be functionalised with radioisotopes without compromising target specificity. The attachment of different radioisotopes can determine the aptamer’s functionality in the treatment of HER2(+) BC. Several HER2 aptamers and investigations of them have been described and evaluated in this paper. We also provide recommendations for future studies with HER2 aptamers to target HER2(+) BC.

Genetic Dissection of a Prevalent Plasmid-Encoded Conjugation System in Lactococcus lactis

Plasmid pNP40, which was first identified nearly 40 years ago in Lactococcus lactis subsp. lactis biovar diacetylactis DRC3, encodes functions such as heavy metal-, bacteriophage-, and nisin-resistance, as well as plasmid transfer ability by conjugation. Here, we report an optimized conjugation protocol for this plasmid, yielding a transfer frequency that is approximately 4,000-fold higher than those previously reported in literature, while we also observed high-frequency plasmid co-mobilization. Individual mutations in 18 genes that encompass the presumed conjugation cluster of pNP40 were generated using ssDNA recombineering to evaluate the role of each gene in the conjugation process. A possible transcriptional repressor of this conjugation cluster, the product of the traR gene, was identified in this manner. This mutational analysis, paired with bioinformatic predictions as based on sequence and structural similarities, allowed us to generate a preliminary model of the pNP40 conjugation machinery.

State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis

Antibody-drug conjugates (ADCs) are biotherapeutics consisting of a tumor-targeting monoclonal antibody (mAb) linked covalently to a cytotoxic drug. Early generation ADCs were predominantly obtained through non-selective conjugation methods based on lysine and cysteine residues, resulting in heterogeneous populations with varying drug-to-antibody ratios (DAR). Site-specific conjugation is one of the current challenges in ADC development, allowing for controlled conjugation and production of homogeneous ADCs. We report here the characterization of a site-specific DAR2 ADC generated with the GlyCLICK three-step process, which involves glycan-based enzymatic remodeling and click chemistry, using state-of-the-art native mass spectrometry (nMS) methods. The conjugation process was monitored with size exclusion chromatography coupled to nMS (SEC-nMS), which offered a straightforward identification and quantification of all reaction products, providing a direct snapshot of the ADC homogeneity. Benefits of SEC-nMS were further demonstrated for forced degradation studies, for which fragments generated upon thermal stress were clearly identified, with no deconjugation of the drug linker observed for the T-GlyGLICK-DM1 ADC. Lastly, innovative ion mobility-based collision-induced unfolding (CIU) approaches were used to assess the gas-phase behavior of compounds along the conjugation process, highlighting an increased resistance of the mAb against gas-phase unfolding upon drug conjugation. Altogether, these state-of-the-art nMS methods represent innovative approaches to investigate drug loading and distribution of last generation ADCs, their evolution during the bioconjugation process and their impact on gas-phase stabilities. We envision nMS and CIU methods to improve the conformational characterization of next generation-empowered mAb-derived products such as engineered nanobodies, bispecific ADCs or immunocytokines.

Multiple Layered Control of the Conjugation Process of the Bacillus subtilis Plasmid pLS20

Bacterial conjugation is the main horizontal gene transfer route responsible for the spread of antibiotic resistance, virulence and toxin genes. During conjugation, DNA is transferred from a donor to a recipient cell via a sophisticated channel connecting the two cells. Conjugation not only affects many different aspects of the plasmid and the host, ranging from the properties of the membrane and the cell surface of the donor, to other developmental processes such as competence, it probably also poses a burden on the donor cell due to the expression of the large number of genes involved in the conjugation process. Therefore, expression of the conjugation genes must be strictly controlled. Over the past decade, the regulation of the conjugation genes present on the conjugative Bacillus subtilis plasmid pLS20 has been studied using a variety of methods including genetic, biochemical, biophysical and structural approaches. This review focuses on the interplay between RcopLS20, RappLS20 and Phr*pLS20, the proteins that control the activity of the main conjugation promoter Pc located upstream of the conjugation operon. Proper expression of the conjugation genes requires the following two fundamental elements. First, conjugation is repressed by default and an intercellular quorum-signaling system is used to sense conditions favorable for conjugation. Second, different layers of regulation act together to repress the Pc promoter in a strict manner but allowing rapid activation. During conjugation, ssDNA is exported from the cell by a membrane-embedded DNA translocation machine. Another membrane-embedded DNA translocation machine imports ssDNA in competent cells. Evidences are reviewed indicating that conjugation and competence are probably mutually exclusive processes. Some of the questions that remain unanswered are discussed.

Konjugasi pada Bahasa Sikka Krove di Kabupaten Sikka Nusa Tenggara Timur

Sikka’s verbs have different features from other regional languages in their application in sentences. This is because the Sikka verbs undergo a conjugation process, which is a change in the form of the verb based on the persona pronoun that precedes it. Based on observation during the language practice of speakers of this language, it can be seen that certain verbs, namely 'eat' and 'drink' in Sikka language, are unique, namely the existence of an inflection process that is more conical in conjugation. This research aims to describe the various patterns formed as a conjugation process. This type of research is qualitative, that the data is a description using verb words. The method used in this research is descriptive qualitative method, namely analyzing descriptive data by describing various conjugation patterns that occur in the Sikka language. The result obtained is a form of conjugation based on the subject followed by the verb "eat" and "drink" in the Sikka language. That it turns out that there is a change in the form of the verb due to a change in the subject. Meanwhile, the time form and other forms were not found in this study.