Unlocking Genetic Mysteries during the Epic Sperm Journey toward Fertilization: Further Expanding Cre Mouse Lines.

The spatiotemporal expression patterns of genes are crucial for maintaining normal physiological functions in animals. Conditional gene knockout using the cyclization recombination enzyme (Cre)/locus of crossover of P1 (Cre/LoxP) strategy has been extensively employed for functional assays at specific tissue or developmental stages. This approach aids in uncovering the associations between phenotypes and gene regulation while minimizing interference among distinct tissues. Various Cre-engineered mouse models have been utilized in the male reproductive system, including Dppa3-MERCre for primordial germ cells, Ddx4-Cre and Stra8-Cre for spermatogonia, Prm1-Cre and Acrv1-iCre for haploid spermatids, Cyp17a1-iCre for the Leydig cell, Sox9-Cre for the Sertoli cell, and Lcn5/8/9-Cre for differentiated segments of the epididymis. Notably, the specificity and functioning stage of Cre recombinases vary, and the efficiency of recombination driven by Cre depends on endogenous promoters with different sequences as well as the constructed Cre vectors, even when controlled by an identical promoter. Cre mouse models generated via traditional recombination or CRISPR/Cas9 also exhibit distinct knockout properties. This review focuses on Cre-engineered mouse models applied to the male reproductive system, including Cre-targeting strategies, mouse model screening, and practical challenges encountered, particularly with novel mouse strains over the past decade. It aims to provide valuable references for studies conducted on the male reproductive system.

Protocol for the establishment of a serine integrase-based platform for functional validation of genetic switch controllers in eukaryotic cells.

Serine integrases (Ints) are a family of site-specific recombinases (SSRs) encoded by some bacteriophages to integrate their genetic material into the genome of a host. Their ability to rearrange DNA sequences in different ways including inversion, excision, or insertion with no help from endogenous molecular machinery, confers important biotechnological value as genetic editing tools with high host plasticity. Despite advances in their use in prokaryotic cells, only a few Ints are currently used as gene editors in eukaryotes, partly due to the functional loss and cytotoxicity presented by some candidates in more complex organisms. To help expand the number of Ints available for the assembly of more complex multifunctional circuits in eukaryotic cells, this protocol describes a platform for the assembly and functional screening of serine-integrase-based genetic switches designed to control gene expression by directional inversions of DNA sequence orientation. The system consists of two sets of plasmids, an effector module and a reporter module, both sets assembled with regulatory components (as promoter and terminator regions) appropriate for expression in mammals, including humans, and plants. The complete method involves plasmid design, DNA delivery, testing and both molecular and phenotypical assessment of results. This platform presents a suitable workflow for the identification and functional validation of new tools for the genetic regulation and reprogramming of organisms with importance in different fields, from medical applications to crop enhancement, as shown by the initial results obtained. This protocol can be completed in 4 weeks for mammalian cells or up to 8 weeks for plant cells, considering cell culture or plant growth time.

Screening Key Sites of Class 2 Integron Integrase that Impact Recombination Efficiency.

By capturing and expressing exogenous resistance gene cassettes through site-specific recombination, integrons play important roles in the horizontal transfer of antimicrobial resistant genes among bacteria. The characteristics of integron integrase make it to be a potential gene editing tool enzyme. In this study, a random mutation library using error-prone PCR was constructed, and amino acid residues mutants that impact on attI2 × attC or attC × attC recombination efficiency were screened and analyzed. Thirteen amino acid mutations were identified to be critical impacted on site-specific recombination of IntI2, including the predicted catalyzed site Y301. Nine of 13 mutated amino acid residues that have critically impacted on IntI2 activity were relative concentrated and near the predicted catalyzed site Y301 in the predicted three-dimensional structure indicated the importance of this area in maintain the activity of IntI2. No mutant with obviously increased recombination activity (more than four-fold as high as that of wild IntI2) was found in library screening, except P95S, R100K slightly increased (within two-fold) the excision activity of IntI2, and S243T slightly increased (within two-fold) both excision and integration activity of IntI2. These findings will provide clues for further specific modification of integron integrase to be a tool enzyme as well as establishing a new gene editing system and applied practically.

A knock-in allele of Hand2 expressing Dre recombinase.

HAND2 is a basic helix-loop-helix transcription factor with diverse functions during development. To facilitate the investigation of genetic and functional diversity among Hand2-expressing cells in the mouse, we have generated Hand2Dre, a knock-in allele expressing Dre recombinase. To avoid disrupting Hand2 function, the Dre cDNA is inserted at the 3' end of the Hand2 coding sequence following a viral 2A peptide. Hand2Dre homozygotes can therefore be used in complex crosses to increase the proportion of useful genotypes among offspring. Dre expression in mid-gestation Hand2Dre embryos is indistinguishable from wild-type Hand2 expression, and HandDre efficiently recombines rox target sites in vivo. In combination with existing Cre and Flp mouse lines, Hand2Dre will therefore extend the ability to perform genetic intersectional labeling, fate mapping, and functional manipulation of subpopulations of cells characterized by developmental expression of Hand2.

Generation of Slco1a4-CreERT2-tdTomato Knock-in Mice for Specific Cerebrovascular Endothelial Cell Targeting.

The cerebrovascular endothelial cells with distinct characteristics line cerebrovascular blood vessels and are the fundamental structure of the blood-brain barrier, which is important for the development and homeostatic maintenance of the central nervous system. Cre-LoxP system-based spatial gene manipulation in mice is critical for investigating the physiological functions of key factors or signaling pathways in cerebrovascular endothelial cells. However, there is a lack of Cre recombinase mouse lines that specifically target cerebrovascular endothelial cells. Here, using a publicly available single-cell RNAseq database, we screened the solute carrier organic anion transporter family member 1a4 (Slco1a4) as a candidate marker of cerebrovascular endothelial cells. Then, we generated an inducible Cre mouse line in which a CreERT2-T2A-tdTomato cassette was placed after the initiation codon ATG of the Slco1a4 locus. We found that tdTomato, which can indicate the endogenous Slco1a4 expression, was expressed in almost all cerebrovascular endothelial cells but not in any other non-endothelial cell types in the brain, including neurons, astrocytes, oligodendrocytes, pericytes, smooth muscle cells, and microglial cells, as well as in other organs. Consistently, when crossing the ROSA26LSL-EYFP Cre reporter mouse, EYFP also specifically labeled almost all cerebrovascular endothelial cells upon tamoxifen induction. Overall, we generated a new inducible Cre line that specifically targets cerebrovascular endothelial cells.

Uncoupling protein 1-driven Cre (Ucp1-Cre) is expressed in the epithelial cells of mammary glands and various non-adipose tissues.

OBJECTIVE: Uncoupling protein 1 (UCP1), a mitochondrial protein responsible for nonshivering thermogenesis in adipose tissue, serves as a distinct marker for thermogenic brown and beige adipocytes. Ucp1-Cre mice are thus widely used to genetically manipulate these thermogenic adipocytes. However, evidence suggests that UCP1 may also be expressed in non-adipocyte cell types. In this study, we investigated the presence of UCP1 expression in different mouse tissues that have not been previously reported. METHODS: We employed Ucp1-Cre mice crossed with Cre-inducible transgenic reporter Nuclear tagging and Translating Ribosome Affinity Purification (NuTRAP) mice to investigate Ucp1-Cre expression in various tissues of adult female mice and developing embryos. Tamoxifen-inducible Ucp1-CreERT2 mice crossed with NuTRAP mice were used to assess active Ucp1 expression in adult mice. Immunostaining, RNA analysis, and single-cell/nucleus RNA-seq (sc/snRNA-seq) data analysis were performed to determine the expression of endogenous UCP1 and Ucp1-Cre-driven reporter expression. We also investigated the impact of UCP1 deficiency on mammary gland development and function using Ucp1-knockout (KO) mice. RESULTS: Ucp1-Cre expression was observed in the mammary glands within the inguinal white adipose tissue of female Ucp1-Cre; NuTRAP mice. Ucp1-Cre was activated during embryonic development in various tissues, including mammary glands, as well as in the brain, kidneys, eyes, and ears, specifically in epithelial cells in these organs. However, Ucp1-CreERT2 showed no or only partial activation in these tissues of adult mice, indicating the potential for low or transient expression of endogenous Ucp1. While sc/snRNA-seq data suggest potential expression of UCP1 in mammary epithelial cells in adult mice and humans, Ucp1-KO female mice displayed normal mammary gland development and function. CONCLUSIONS: Our findings reveal widespread Ucp1-Cre expression in various non-adipose tissue types, starting during early development. These results highlight the importance of exercising caution when interpreting data and devising experiments involving Ucp1-Cre mice.

ORBIT for E. coli: kilobase-scale oligonucleotide recombineering at high throughput and high efficiency.

Microbiology and synthetic biology depend on reverse genetic approaches to manipulate bacterial genomes; however, existing methods require molecular biology to generate genomic homology, suffer from low efficiency, and are not easily scaled to high throughput. To overcome these limitations, we developed a system for creating kilobase-scale genomic modifications that uses DNA oligonucleotides to direct the integration of a non-replicating plasmid. This method, Oligonucleotide Recombineering followed by Bxb-1 Integrase Targeting (ORBIT) was pioneered in Mycobacteria, and here we adapt and expand it for Escherichia coli. Our redesigned plasmid toolkit for oligonucleotide recombineering achieved significantly higher efficiency than λ Red double-stranded DNA recombineering and enabled precise, stable knockouts (≤134 kb) and integrations (≤11 kb) of various sizes. Additionally, we constructed multi-mutants in a single transformation, using orthogonal attachment sites. At high throughput, we used pools of targeting oligonucleotides to knock out nearly all known transcription factor and small RNA genes, yielding accurate, genome-wide, single mutant libraries. By counting genomic barcodes, we also show ORBIT libraries can scale to thousands of unique members (>30k). This work demonstrates that ORBIT for E. coli is a flexible reverse genetic system that facilitates rapid construction of complex strains and readily scales to create sophisticated mutant libraries.

Differential Impact In Vivo of Pf4-ΔCre-Mediated and Gp1ba-ΔCre-Mediated Depletion of Cyclooxygenase-1 in Platelets in Mice.

BACKGROUND: Low-dose aspirin is widely used for the secondary prevention of cardiovascular disease. The beneficial effects of low-dose aspirin are attributable to its inhibition of platelet Cox (cyclooxygenase)-1-derived thromboxane A2. Until recently, the use of the Pf4 (platelet factor 4) Cre has been the only genetic approach to generating megakaryocyte/platelet ablation of Cox-1 in mice. However, Pf4-ΔCre displays ectopic expression outside the megakaryocyte/platelet lineage, especially during inflammation. The use of the Gp1ba (glycoprotein 1bα) Cre promises a more specific, targeted approach. METHODS: To evaluate the role of Cox-1 in platelets, we crossed Pf4-ΔCre or Gp1ba-ΔCre mice with Cox-1flox/flox mice to generate platelet Cox-1-/- mice on normolipidemic and hyperlipidemic (Ldlr-/-; low-density lipoprotein receptor) backgrounds. RESULTS: Ex vivo platelet aggregation induced by arachidonic acid or adenosine diphosphate in platelet-rich plasma was inhibited to a similar extent in Pf4-ΔCre Cox-1-/-/Ldlr-/- and Gp1ba-ΔCre Cox-1-/-/Ldlr-/- mice. In a mouse model of tail injury, Pf4-ΔCre-mediated and Gp1ba-ΔCre-mediated deletions of Cox-1 were similarly efficient in suppressing platelet prostanoid biosynthesis. Experimental thrombogenesis and attendant blood loss were similar in both models. However, the impact on atherogenesis was divergent, being accelerated in the Pf4-ΔCre mice while restrained in the Gp1ba-ΔCres. In the former, accelerated atherogenesis was associated with greater suppression of PGI2 biosynthesis, a reduction in the lipopolysaccharide-evoked capacity to produce PGE2 (prostaglandin E) and PGD2 (prostanglandin D), activation of the inflammasome, elevated plasma levels of IL-1ß (interleukin), reduced plasma levels of HDL-C (high-density lipoprotein receptor-cholesterol), and a reduction in the capacity for reverse cholesterol transport. By contrast, in the latter, plasma HDL-C and α-tocopherol were elevated, and MIP-1α (macrophage inflammatory protein-1α) and MCP-1 (monocyte chemoattractant protein 1) were reduced. CONCLUSIONS: Both approaches to Cox-1 deletion similarly restrain thrombogenesis, but a differential impact on Cox-1-dependent prostanoid formation by the vasculature may contribute to an inflammatory phenotype and accelerated atherogenesis in Pf4-ΔCre mice.

Belt and braces: Two escape ways to maintain the cassette reservoir of large chromosomal integrons.

Integrons are adaptive devices that capture, stockpile, shuffle and express gene cassettes thereby sampling combinatorial phenotypic diversity. Some integrons called sedentary chromosomal integrons (SCIs) can be massive structures containing hundreds of cassettes. Since most of these cassettes are non-expressed, it is not clear how they remain stable over long evolutionary timescales. Recently, it was found that the experimental inversion of the SCI of Vibrio cholerae led to a dramatic increase of the cassette excision rate associated with a fitness defect. Here, we question the evolutionary sustainability of this apparently counter selected genetic context. Through experimental evolution, we find that the integrase is rapidly inactivated and that the inverted SCI can recover its original orientation by homologous recombination between two insertion sequences (ISs) present in the array. These two outcomes of SCI inversion restore the normal growth and prevent the loss of cassettes, enabling SCIs to retain their roles as reservoirs of functions. These results illustrate a nice interplay between gene orientation, genome rearrangement, bacterial fitness and demonstrate how integrons can benefit from their embedded ISs.

The generation and characterization of a transgenic zebrafish line with lens-specific Cre expression.

Purpose: Danio rerio zebrafish constitute a popular model for studying lens development and congenital cataracts. However, the specific deletion of a gene with a Cre/LoxP system in the zebrafish lens is unavailable because of the lack of a lens-Cre-transgenic zebrafish. This study aimed to generate a transgenic zebrafish line in which Cre recombinase was specifically expressed in the lens. Methods: The pTol2 cryaa:Cre-polyA-cryaa:EGFP (enhanced green fluorescent protein) plasmid was constructed and co-injected with Tol2-transposase into one-to-two-cell-stage wild-type (WT) zebrafish embryos. Whole-mount in situ hybridization (ISH), tissue section, hematoxylin and eosin staining, a Western blot, a split-lamp observation, and a grid transmission assay were used to analyze the Cre expression, lens structure, and lens transparency of the transgenic zebrafish. Results: In this study, we generated a transgenic zebrafish line, zTg(cryaa:Cre-cryaa:EGFP), in which Cre recombinase and EGFP were driven by the lens-specific cryaa promoter. zTg(cryaa:Cre-cryaa:EGFP) began to express Cre and EGFP specifically in the lens at the 22 hpf stage, and this ectopic Cre could efficiently and specifically delete the red fluorescent protein (RFP) signal from the lens when zTg(cryaa:Cre-cryaa:EGFP) embryos were injected with the loxP-flanked RFP plasmid. The overexpression of Cre and EGFP did not impair zebrafish development or lens transparency. Accordingly, this zTg(cryaa:Cre-cryaa:EGFP) zebrafish line is a useful tool for gene editing, specifically with zebrafish lenses. Conclusions: We established a zTg(cryaa:Cre-cryaa:EGFP) zebrafish line that can specifically express an active Cre recombinase in lens tissues. This transgenic zebrafish line can be used as a tool to specifically manipulate a gene in zebrafish lenses.

Complementary and Inducible creERT2 Mouse Models for Functional Evaluation of Endothelial Cell Subtypes in the Bone Marrow.

In the adult bone marrow (BM), endothelial cells (ECs) are an integral component of the hematopoietic stem cell (HSC)-supportive niche, which modulates HSC activity by producing secreted and membrane-bound paracrine signals. Within the BM, distinct vascular arteriole, transitional, and sinusoidal EC subtypes display unique paracrine expression profiles and create anatomically-discrete microenvironments. However, the relative contributions of vascular endothelial subtypes in supporting hematopoiesis is unclear. Moreover, constitutive expression and off-target activity of currently available endothelial-specific and endothelial-subtype-specific murine cre lines potentially confound data analysis and interpretation. To address this, we describe two tamoxifen-inducible cre-expressing lines, Vegfr3-creERT2 and Cx40-creERT2, that efficiently label sinusoidal/transitional and arteriole endothelium respectively in adult marrow, without off-target activity in hematopoietic or perivascular cells. Utilizing an established mouse model in which cre-dependent recombination constitutively-activates MAPK signaling within adult endothelium, we identify arteriole ECs as the driver of MAPK-mediated hematopoietic dysfunction. These results define complementary tamoxifen-inducible creERT2-expressing mouse lines that label functionally-discrete and non-overlapping sinusoidal/transitional and arteriole EC populations in the adult BM, providing a robust toolset to investigate the differential contributions of vascular subtypes in maintaining hematopoietic homeostasis.

An Hsp70 promoter-based mouse for heat shock-induced gene modulation.

Physical therapy is extensively employed in clinical settings. Nevertheless, the absence of suitable animal models has resulted in an incomplete understanding of the in vivo mechanisms and cellular distribution that respond to physical stimuli. The objective of this research was to create a mouse model capable of indicating the cells affected by physical stimuli. In this study, we successfully established a mouse line based on the heat shock protein 70 (Hsp70) promoter, wherein the expression of CreERT2 can be induced by physical stimuli. Following stimulation of the mouse tail, ear, or cultured calvarias with heat shock (generated by heating, ultrasound, or laser), a distinct Cre-mediated excision was observed in cells stimulated by these physical factors with minimal occurrence of leaky reporter expression. The application of heat shock to Hsp70-CreERT2; FGFR2-P253R double transgenic mice or Hsp70-CreERT2 mice infected with AAV-BMP4 at calvarias induced the activation of Cre-dependent mutant FGFR2-P253R or BMP4 respectively, thereby facilitating the premature closure of cranial sutures or the repair of calvarial defects. This novel mouse line holds significant potential for investigating the underlying mechanisms of physical therapy, tissue repair and regeneration, lineage tracing, and targeted modulation of gene expression of cells in local tissue stimulated by physical factor at the interested time points. KEY MESSAGES: In the study, an Hsp70-CreERT2 transgenic mouse was generated for heat shock-induced gene modulation. Heat shock, ultrasound, and laser stimulation effectively activated Cre expression in Hsp70-CreERT2; reporter mice, which leads to deletion of floxed DNA sequence in the tail, ear, and cultured calvaria tissues of mice. Local laser stimuli on cultured calvarias effectively induce Fgfr2-P253R expression in Hsp70-mTmG-Fgfr2-P253R mice and result in accelerated premature closure of cranial suture. Heat shock activated AAV9-FLEX-BMP4 expression and subsequently promoted the repair of calvarial defect of Hsp70-CreERT2; Rosa26-mTmG mice.

Characterization and genome-informatic analysis of a novel lytic mendocina phage vB_PmeS_STP12 suitable for phage therapy pseudomonas or biocontrol.

BACKGROUND: A novel lytic bacteriophage (phage) was isolated with Pseudomonas mendocina strain STP12 (P. mendocina) from the untreated site of Sewage Treatment Plant of Lovely Professional University, India. P. mendocina is a Gram-negative, rod-shaped, aerobic bacterium belonging to the family Pseudomonadaceae and has been reported in fifteen (15) cases of economically important diseases worldwide. METHODS AND RESULTS: Here, a novel phage specifically infecting and killing P. mendocina strain STP12 was isolated from sewage sample using enrichment, spot test and double agar overlay (DAOL) method and was designated as vB_PmeS_STP12. The phage vB-PmeS-STP12 was viable at wide range of pH and temperature ranging from 4 to10 and - 20 to 70 °C respectively. Host range and efficiency of plating (EOP) analysis indicated that phage vB-PmeS-STP12 was capable of infecting and killing P. mendocina strain STP6 with EOP of 0.34. Phage vB_PmeS_STP12 was found to have a significant bacterial reduction (p < 0.005) at all the doses administered, particularly at optimal MOI of 1 PFU/CFU, compared to the control. Morphological analysis using high resolution transmission electron microscopy (HR-TEM) revealed an icosahedral capsid of ~ 55 nm in diameter on average with a short, non-contractile tail. The genome of vB_PmeS_STP12 is a linear, dsDNA containing 36,212 bp in size with a GC content of 58.87% harbouring 46 open reading frames (ORFs). The 46 predicted ORFs encode proteins with functional information categorized as lysis, replication, packaging, regulation, assembly, infection, immune, and hypothetical. However, the genome of vB_PmeS_STP12 appeared to be devoid of tRNAs, integrase gene, toxins genes, virulence factors, antimicrobial resistance genes (ARGs) and CRISPR arrays. The blast analysis with phylogeny revealed that vB_PmeS_STP12 is genetically similar to Pseudomonas phage PMBT14, Pseudomonas phage Almagne and Serratia phage Serbin with a highest identity of 74.00%, 74.93% and 59.48% respectively. CONCLUSIONS: Taken together, characterization, morphological analysis and genome-informatics indicated that vB_PmeS_STP12 is podovirus morphotype belonging to the class Caudoviticetes, family Zobellviridae which appeared to be devoid of integrase gene, ARGs, CRISPR arrays, virulence factors and toxins genes, exhibiting stability and infectivity at wide range of pH (4 to10) and temperature (-20 to 70 °C), thereby making vB_PmeS_STP12 suitable for phage therapy or biocontrol. Based on the bibliometric analysis and data availability with respect to sequences deposited in GenBank, this is the first report of a phage infecting Pseudomonas mendocina.

Glycosylation as a tracer of off-target Cre-lox activation in development.

The Cre-lox system is one of the most widely used methods for lineage-specific and inducible genome editing in vivo. However, incomplete penetrance and off-target effects due to transient promoter expression in a stem or pluripotent precursor cell can be problematic and difficult to detect, especially if the target gene is not normally present in the fully differentiated but off-target cells. Yet, the loss of the target gene through the transient expression of Cre may impact the differentiation of those cells by virtue of transient expression in a precursor population. In these situations, off-target effects in an unknown precursor cell can, at best, complicate conclusions drawn from the model, and at worst, invalidate all data generated from that knockout strain. Thus, identifying Cre-driver promoter expression along entire cell lineages is crucial to improve rigor and reproducibility. As an example, transient expression in an early precursor cell has been documented in a variety of Cre strains such as the Tie2-based Cre-driver system that is used as an "endothelial cell-specific" model 1. Yet, Tie2 is now known to be transiently expressed in a stem cell upstream of both hematopoietic and endothelial cell lineages. Here, we use the Tie2 Cre-driver strain to demonstrate that due to its ubiquitous nature, plasma membrane glycans are a useful marker of both penetrance and specificity of a Cre-based knockout.

Albumin promoter-driven FlpO expression induces efficient genetic recombination in mouse liver.

Tissue-specific gene manipulations are widely used in genetically engineered mouse models. A single recombinase system, such as the one using Alb-Cre, has been commonly used for liver-specific genetic manipulations. However, most diseases are complex, involving multiple genetic changes and various cell types. A dual recombinase system is required for conditionally modifying different genes sequentially in the same cell or inducing genetic changes in different cell types within the same organism. A FlpO cDNA was inserted between the last exon and 3'-UTR of the mouse albumin gene in a bacterial artificial chromosome (BAC-Alb-FlpO). The founders were crossed with various reporter mice to examine the efficiency of recombination. Liver cancer tumorigenesis was investigated by crossing the FlpO mice with FSF-KrasG12D mice and p53frt mice (KPF mice). BAC-Alb-FlpO mice exhibited highly efficient recombination capability in both hepatocytes and intrahepatic cholangiocytes. No recombination was observed in the duodenum and pancreatic cells. BAC-Alb-FlpO-mediated liver-specific expression of mutant KrasG12D and conditional deletion of p53 gene caused the development of liver cancer. Remarkably, liver cancer in these KPF mice manifested a distinctive mixed hepatocellular carcinoma and cholangiocarcinoma phenotype. A highly efficient and liver-specific BAC-Alb-FlpO mouse model was developed. In combination with other Cre lines, different genes can be manipulated sequentially in the same cell, or distinct genetic changes can be induced in different cell types of the same organism.NEW & NOTEWORTHY A liver-specific Alb-FlpO mouse line was generated. By coupling it with other existing CreERT or Cre lines, the dual recombinase approach can enable sequential gene modifications within the same cell or across various cell types in an organism for liver research through temporal and spatial gene manipulations.

Comparison of Integrase Strand Transfer Inhibitors (INSTIs) and Protease-Boosted Inhibitors (PIs) on the Reduction in Chronic Immune Activation in a Virally Suppressed, Mainly Male Population Living with HIV (PLWH).

Background and Objectives: The success of combined antiretroviral therapy (cART) has led to a dramatic improvement in the life expectancy of people living with HIV (PLWH). However, there has been an observed increase in cardiometabolic, bone, renal, hepatic, and neurocognitive manifestations, as well as neoplasms, known as serious non-AIDS events/SNAEs, compared to the general population of corresponding age. This increase is linked to a harmful phenomenon called inflammaging/immunosenescence, which is driven by chronic immune activation and intestinal bacterial translocation. In this study, we examined immunological and metabolic parameters in individuals receiving current cART. Materials and Methods: The study was conducted at Laiko General Hospital in Athens, Greece. Plasma concentrations of sCD14, IL-6, SuPAR, I-FABP, and LBP were measured in virally suppressed PLWH under cART with at least 350 CD4 lymphocytes/µL. We compared these levels between PLWH receiving integrase strand transfer inhibitors (INSTIs) and protease inhibitors (PIs) and attempted to correlate them with chronic immune activation and metabolic parameters. Results: Data from 28 PLWH were analyzed, with a mean age of 52 and 93% being males. Among the two comparison groups, IL-6 levels were higher in the PIs group (5.65 vs. 7.11 pg/mL, p = 0.03). No statistically significant differences were found in the other measured parameters. A greater proportion of PLWH under INSTIs had normal-range LBP (33% vs. 0%, p = 0.04). When using inverse probability of treatment weighting, no statistically significant differences in the measured parameters were found between the two groups (sCD14 p = 0.511, IL-6 p = 0.383, SuPAR p = 0.793, I-FABP p = 0.868, and LBP p = 0.663). Glucose levels were found to increase after viral suppression in the entire sample (92 mg/dL vs. 98 mg/dL, p = 0.009). Total (191 mg/dL vs. 222 mg/dL, p = 0.005) and LDL cholesterol (104 mg/dL vs. 140 mg/dL, p = 0.002) levels were higher in the PIs group. No significant differences were observed in liver and renal function tests. Conclusions: Further investigation is warranted for PLWH on cART-containing INSTI regimens to explore potential reductions in chronic immune activation and intestinal bacterial translocation.

A synthetic differentiation circuit in Escherichia coli for suppressing mutant takeover.

Differentiation is crucial for multicellularity. However, it is inherently susceptible to mutant cells that fail to differentiate. These mutants outcompete normal cells by excessive self-renewal. It remains unclear what mechanisms can resist such mutant expansion. Here, we demonstrate a solution by engineering a synthetic differentiation circuit in Escherichia coli that selects against these mutants via a biphasic fitness strategy. The circuit provides tunable production of synthetic analogs of stem, progenitor, and differentiated cells. It resists mutations by coupling differentiation to the production of an essential enzyme, thereby disadvantaging non-differentiating mutants. The circuit selected for and maintained a positive differentiation rate in long-term evolution. Surprisingly, this rate remained constant across vast changes in growth conditions. We found that transit-amplifying cells (fast-growing progenitors) underlie this environmental robustness. Our results provide insight into the stability of differentiation and demonstrate a powerful method for engineering evolutionarily stable multicellular consortia.

The effect of a treatment switch to integrase Strand transfer inhibitor-based regimens on weight gain and other metabolic syndrome-related conditions.

OBJECTIVE: This study aimed to assess weight gain associated with treatment switching to INSTI-based regimens in people living with HIV (PLWH) and to determine whether it is accompanied by worsening features of hypertension, dyslipidemia, or hyperglycemia. METHODS: In this two-center retrospective observational study, we assessed weight gain and metabolic features in PLWH who switched to an INSTI-based regimen (study group) as compared to patients who remained on a non-INSTI regimen (control group) over a 24-month follow-up period. RESULTS: One-hundred seventy-four PLWH were included in the study group, and 175 were included in the control group. The study group gained 2.51 kg ± 0.31 (mean ± standard deviation) over the 2 years of follow-up, while the control group gained 1.1 ± 0.31 kg over the same time course (p < 0.001). INSTI treatment, Caucasian origin, and lower BMI were risk factors associated with excessive weight gain during the 2 years of follow-up. Among metabolic parameters, only glucose levels increased after initiating INSTI-based regimens, although limited to males of African origin (p = 0.009). CONCLUSIONS: We observed a mild weight gain after switching to INSTI-based regimens, with no major impact on metabolic parameters over 2 years of follow-up. Longer follow-up might be needed to observe the adverse metabolic effects of INSTI-based regimens. The impact on weight gain should be discussed with every patient before the treatment switch to ensure a balanced diet and physical activity to prevent excessive weight gain that might hamper compliance with ART.

The effect of site-specific recombinases XerCD on the removal of over-replicated chromosomal DNA through outer membrane vesicles in bacteria.

Outer membrane vesicles (OMVs) are universally produced by Gram-negative bacteria and play important roles in symbiotic and pathogenic interactions. The DNA from the lumen of OMVs from the Alphaproteobacterium Dinoroseobacter shibae was previously shown to be enriched for the region around the terminus of replication ter and specifically for the recognition sequence dif of the two site-specific recombinases XerCD. These enzymes are highly conserved in bacteria and play an important role in the last phase of cell division. Here, we show that a similar enrichment of ter and dif is found in the DNA inside OMVs from Prochlorococcus marinus, Pseudomonas aeruginosa, Vibrio cholerae, and Escherichia coli. The deletion of xerC or xerD in E. coli reduced the enrichment peak directly at the dif sequence, while the enriched DNA region around ter became broader, demonstrating that either enzyme influences the DNA content inside the lumen of OMVs. We propose that the intra-vesicle DNA originated from over-replication repair and the XerCD enzymes might play a role in this process, providing them with a new function in addition to resolving chromosome dimers.IMPORTANCEImprecise termination of replication can lead to over-replicated parts of bacterial chromosomes that have to be excised and removed from the dividing cell. The underlying mechanism is poorly understood. Our data show that outer membrane vesicles (OMVs) from diverse Gram-negative bacteria are enriched for DNA around the terminus of replication ter and the site-specific XerCD recombinases influence this enrichment. Clearing the divisome from over-replicated parts of the bacterial chromosome might be a so far unrecognized and conserved function of OMVs.

Recycling selectable markers via Cre/loxP system for constructing Komagataella phaffii strains co-expressing multiple proteins.

OBJECTIVE: A convenient strategy was developed to recycle selectable markers using Cre/loxP system for constructing Komagataella phaffii strains co-expressing multiple proteins. RESULTS: A plasmid in this strategy was generated from pPICZαA with integration of lox71-Sh ble-lox66. Firstly, the plasmid was inserted with one target protein gene and then transformed into K. phaffii KM71. Secondly, the auxiliary plasmid pPICZαA/cre/his4 containing CRE recombinase gene was further chromosomally inserted to Sh ble gene therein. Finally, methanol induction was conducted to produce CRE for Cre/loxP-mediated recombination, and consequently, the sequence between lox71 and lox66 was deleted, leading to recycling of ZeoR and His- markers. Then the resulted strain expressing the one target protein was used as the host to which another target protein gene could be inserted by the same procedures. CONCLUSIONS: With easy manipulation, the method was effective in recycling of the selectable markers, and consequently two protein genes were sequential integrated chromosomally and successfully co-expressed in the yeast.