Delivery of DNA-Based Therapeutics for Treatment of Chronic Diseases.

Gene therapy and its role in the medical field have evolved drastically in recent decades. Studies aim to define DNA-based medicine as well as encourage innovation and the further development of novel approaches. Gene therapy has been established as an alternative approach to treat a variety of diseases. Its range of mechanistic applicability is wide; gene therapy has the capacity to address the symptoms of disease, the body's ability to fight disease, and in some cases has the ability to cure disease, making it a more attractive intervention than some traditional approaches to treatment (i.e., medicine and surgery). Such versatility also suggests gene therapy has the potential to address a greater number of indications than conventional treatments. Many DNA-based therapies have shown promise in clinical trials, and several have been approved for use in humans. Whereas current treatment regimens for chronic disease often require frequent dosing, DNA-based therapies can produce robust and durable expression of therapeutic genes with fewer treatments. This benefit encourages the application of DNA-based gene therapy to manage chronic diseases, an area where improving efficiency of current treatments is urgent. Here, we provide an overview of two DNA-based gene therapies as well as their delivery methods: adeno associated virus (AAV)-based gene therapy and plasmid DNA (pDNA)-based gene therapy. We will focus on how these therapies have already been utilized to improve treatment of chronic disease, as well as how current literature supports the expansion of these therapies to treat additional chronic indications in the future.

Enhancing In Vivo Electroporation Efficiency through Hyaluronidase: Insights into Plasmid Distribution and Optimization Strategies.

Electroporation (EP) stands out as a promising non-viral plasmid delivery strategy, although achieving optimal transfection efficiency in vivo remains a challenge. A noteworthy advancement in the field of in vivo EP is the application of hyaluronidase, an enzyme with the capacity to degrade hyaluronic acid in the extracellular matrix, which thereby enhances DNA transfer efficiency by 2- to 3-fold. This paper focuses on elucidating the mechanism of hyaluronidase's impact on transfection efficiency. We demonstrate that hyaluronidase promotes a more uniform distribution of plasmid DNA (pDNA) within skeletal muscle. Additionally, our study investigates the effect of the timing of hyaluronidase pretreatment on EP efficiency by including time intervals of 0, 5, and 30 min between hyaluronidase treatment and the application of pulses. Serum levels of the pDNA-encoded transgene reveal a minimal influence of the hyaluronidase pretreatment time on the final serum protein levels following delivery in both mice and rabbit models. Leveraging bioimpedance measurements, we capture morphological changes in muscle induced by hyaluronidase treatment, which result in a varied pDNA distribution. Subsequently, these findings are employed to optimize EP electrical parameters following hyaluronidase treatment in animal models. This paper offers novel insights into the potential of hyaluronidase in enhancing the effectiveness of in vivo EP, as well as guides optimized electroporation strategies following hyaluronidase use.

A nuclear cAMP microdomain suppresses tumor growth by Hippo pathway inactivation.

Cyclic AMP (cAMP) signaling is localized to multiple spatially distinct microdomains, but the role of cAMP microdomains in cancer cell biology is poorly understood. Here, we present a tunable genetic system that allows us to activate cAMP signaling in specific microdomains. We uncover a nuclear cAMP microdomain that activates a tumor-suppressive pathway in a broad range of cancers by inhibiting YAP, a key effector protein of the Hippo pathway, inside the nucleus. We show that nuclear cAMP induces a LATS-dependent pathway leading to phosphorylation of nuclear YAP solely at serine 397 and export of YAP from the nucleus with no change in YAP protein stability. Thus, nuclear cAMP inhibition of nuclear YAP is distinct from other known mechanisms of Hippo regulation. Pharmacologic targeting of specific cAMP microdomains remains an untapped therapeutic approach for cancer; thus, drugs directed at the nuclear cAMP microdomain may provide avenues for the treatment of cancer.

Opioid-like peptides and ghrelin mitigation of bariatric results depends on obesity level.

INTRODUCTION: Bariatric surgery, as the only effective treatment of obesity, has strong effects on the metabolism, and nervous and endocrine systems. Thus, based on the different opinions about the efficaciousness of morbid obesity treatments, the aim of the present study was to estimate the association of serum ghrelin and Met-enkephalin (native, five amino acids and cryptic, precursor of enkephalin) concentrations with body mass index (BMI) value in bariatric patients within 30 postoperative days. MATERIAL AND METHODS: The study was performed on 38 female patients divided into two groups: I - BMI lower than 40 kg/m² (n = 18) and II - BMI higher than 40 kg/m² (n = 20). Blood was taken before (-24 h), and 72 h and 30 days after the sleeve gastrectomy. Routine haematological, anthropometric, and metabolic parameters as well as thyroid-stimulating hormone (TSH), ghrelin, and Met-enkephalin values were measured in all patients. RESULTS: There were statistically significant differences between the two groups before the surgery in terms of TSH, both forms of Metenkephalin, triglycerides concentrations, and activity of alanine transaminase (ALT), gamma glutamyltransferase (GGTP), and C-reactive protein (CRP). After 72 h, the serum levels of cryptic Met-enkephalin and CRP, and activity of enzymes varied between the two groups of patients. Thirty days after the surgery, some metabolic and immune parameters were still different in both female groups in favour of patients with lover BMI. However, significant differences were noticed in the levels of ghrelin (increase), and native (decrease) and cryptic Met-enkephalins (increase). CONCLUSIONS: The activity of endogenous peptides in bariatric patients is connected with the degree of obesity. Ghrelin level increases are negatively correlated with native Met-enkephalin changes shortly after bariatric surgery. The interplay of ghrelin and opioids might be considered as a predictor of postoperative weight loss success.

Nucleoplasmic Reticulum Formation in Human Endometrial Cells is Steroid Hormone Responsive and Recruits Nascent Components.

The nuclei of cells may exhibit invaginations of the nuclear envelope under a variety of conditions. These invaginations form a branched network termed the nucleoplasmic reticulum (NR), which may be found in cells in pathological and physiological conditions. While an extensive NR is a hallmark of cellular senescence and shows associations with some cancers, very little is known about the formation of NR in physiological conditions, despite the presence of extensive nuclear invaginations in some cell types such as endometrial cells. Here we show that in these cells the NR is formed in response to reproductive hormones. We demonstrate that oestrogen and progesterone are sufficient to induce NR formation and that this process is reversible without cell division upon removal of the hormonal stimulus. Nascent lamins and phospholipids are incorporated into the invaginations suggesting that there is a dedicated machinery for its formation. The induction of NR in endometrial cells offers a new model to study NR formation and function in physiological conditions.

Formation of a nucleoplasmic reticulum requires de novo assembly of nascent phospholipids and shows preferential incorporation of nascent lamins.

Structure of interphase cell nuclei remains dynamic and can undergo various changes of shape and organisation, in health and disease. The double-membraned envelope that separates nuclear genetic material from the rest of the cell frequently includes deep, branching tubular invaginations that form a dynamic nucleoplasmic reticulum (NR). This study addresses mechanisms by which NR can form in interphase nuclei. We present a combination of Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS) approach and light microscopy techniques to follow formation of NR by using pulse-chase experiments to examine protein and lipid delivery to nascent NR in cultured cells. Lamina protein incorporation was assessed using precursor accumulation (for lamin A) or a MAPLE3 photoconvertible tag (for lamin B1) and membrane phospholipid incorporation using stable isotope labelling with deuterated precursors followed by high resolution NanoSIMS. In all three cases, nascent molecules were selectively incorporated into newly forming NR tubules; thus strongly suggesting that NR formation is a regulated process involving a focal assembly machine, rather than simple physical perturbation of a pre-existing nuclear envelope.

Stress-induced release of Oct-1 from the nuclear envelope is mediated by JNK phosphorylation of lamin B1.

The nuclear lamina can bind and sequester transcription factors (TFs), a function lost if the lamina is abnormal, with missing or mutant lamin proteins. We now show that TF sequestration is not all-or-nothing, but a dynamic physiological response to external signals. We show that the binding of the ubiquitous TF, Oct-1, to lamin B1 was reversed under conditions of cellular stress caused, inter alia, by the chemical methylating agent methylmethanesulfonate (MMS). A search for lamin B1 post-translational modifications that might mediate changes in Oct-1 binding using kinase inhibitors uncovered a role for c-Jun N-terminal kinase (JNK). Phosphoproteomic and site-directed mutagenesis analyses of lamin B1 isolated from control and MMS-treated nuclei identified T575 as a JNK site phosphorylated after stress. A new phospho-T575 specific anti-peptide antibody confirmed increased interphase cellular T575 phosphorylation after cell exposure to certain stress conditions, enabling us to conclude that lamin B1 acts as an interphase kinase target, releasing Oct-1 to execute a protective response to stress.

Results of the Treatment of Infected Nonunions of the Lower Limbs Using the Ilizarov Method.

BACKGROUND: Infected nonunion is a complex complication of the treatment of long bone fractures. An in creased incidence of injuries, including high energy injuries (often open ones), contributes to a higher incidence of nonunion. These primarily infected injuries cause osteomyelitis, which prevents bone union, resulting in an infected nonunion. The Ilizarov method meets the biological and biomechanical treatment requirements, opti mising the process of inflammation healing and producing bone union. MATERIAL AND METHODS: A total of 54 patients were treated in 2000-2014 for nonunion in the lower limbs with the Ilizarov method, which was used after previous treatment had failed. The subjects underwent intra operative resection of the locus of infection, sequestrectomy and a Judet procedure, followed by the use of the Ilizarov apparatus and bone transport, depending on the defect. RESULTS: Inflammation healed in 52 patients (96%) and bone union was achieved in 46 patients (86%). Good outcomes with healed inflammation and bone union were reported in 76% of the cases, fair outcomes with tem porary elimination of the inflammation and without bone union in 16%, and poor outcomes without inflam ma tion healing and without bone union in 7%. CONCLUSIONS: Treatment of infected nonunion can only be effective after eliminating endogenous inflamma tory foci, covering skin defects, ensuring a good condition of the skin and soft tissues, restoring normal blood supply to the bone fragments, and good biomechanical fixation of the fragments with dynamisation or compression. The Ilizarov method is a method of choice in the treatment of cases of infected nonunion where other treatments have failed.

Nanoscale mapping of newly-synthesised phospholipid molecules in a biological cell using tip-enhanced Raman spectroscopy.

Nanoscale chemical mapping of newly-synthesised phospholipid molecules inside a mammalian cell is demonstrated using tip-enhanced Raman spectroscopy (TERS) for the first time using mouse pre-adipocyte cells as a model system. Newly-synthesised membrane phospholipid distribution within a pre-adipocyte cell is mapped with <20 nm spatial resolution, overcoming the diffraction limit of confocal Raman spectroscopy via plasmonic enhancement of Raman signals at a TERS tip-apex.

Shared mechanisms in physiological and pathological nucleoplasmic reticulum formation.

The mammalian nuclear envelope (NE) can develop complex dynamic membrane-bounded invaginations in response to both physiological and pathological stimuli. Since the formation of these nucleoplasmic reticulum (NR) structures can occur during interphase, without mitotic NE breakdown and reassembly, some other mechanism must drive their development. Here we consider models for deformation of the interphase NE, together with the evidence for their potential roles in NR formation.

Methods for Single-Cell Pulse-Chase Analysis of Nuclear Components.

Pulse-chase methods offer powerful tools for following the evolution of a biological system over time, but are usually limited to ensemble measurements of the average behavior of very large numbers of cells. Here we describe three methods ranging from a true pulse-chase, through selective regional photoactivation, to pharmacological induction of an altered protein state, which can be applied to time-dependent studies at the single-cell level. These methods are exemplified by experimental protocols to follow region-selective nuclear envelope targeting of nascent phospholipids, a nascent nuclear lamin protein (lamin B1), and an immature lamin precursor (prelamin A).

Differentially expressed, variant U1 snRNAs regulate gene expression in human cells.

Human U1 small nuclear (sn)RNA, required for splicing of pre-mRNA, is encoded by genes on chromosome 1 (1p36). Imperfect copies of these U1 snRNA genes, also located on chromosome 1 (1q12-21), were thought to be pseudogenes. However, many of these "variant" (v)U1 snRNA genes produce fully processed transcripts. Using antisense oligonucleotides to block the activity of a specific vU1 snRNA in HeLa cells, we have identified global transcriptome changes following interrogation of the Affymetrix Human Exon ST 1.0 array. Our results indicate that this vU1 snRNA regulates expression of a subset of target genes at the level of pre-mRNA processing. This is the first indication that variant U1 snRNAs have a biological function in vivo. Furthermore, some vU1 snRNAs are packaged into unique ribonucleoproteins (RNPs), and many vU1 snRNA genes are differentially expressed in human embryonic stem cells (hESCs) and HeLa cells, suggesting developmental control of RNA processing through expression of different sets of vU1 snRNPs.

Novel non-specific DNA adenine methyltransferases.

The mom gene of bacteriophage Mu encodes an enzyme that converts adenine to N(6)-(1-acetamido)-adenine in the phage DNA and thereby protects the viral genome from cleavage by a wide variety of restriction endonucleases. Mu-like prophage sequences present in Haemophilus influenzae Rd (FluMu), Neisseria meningitidis type A strain Z2491 (Pnme1) and H. influenzae biotype aegyptius ATCC 11116 do not possess a Mom-encoding gene. Instead, at the position occupied by mom in Mu they carry an unrelated gene that encodes a protein with homology to DNA adenine N(6)-methyltransferases (hin1523, nma1821, hia5, respectively). Products of the hin1523, hia5 and nma1821 genes modify adenine residues to N(6)-methyladenine, both in vitro and in vivo. All of these enzymes catalyzed extensive DNA methylation; most notably the Hia5 protein caused the methylation of 61% of the adenines in λ DNA. Kinetic analysis of oligonucleotide methylation suggests that all adenine residues in DNA, with the possible exception of poly(A)-tracts, constitute substrates for the Hia5 and Hin1523 enzymes. Their potential 'sequence specificity' could be summarized as AB or BA (where B = C, G or T). Plasmid DNA isolated from Escherichia coli cells overexpressing these novel DNA methyltransferases was resistant to cleavage by many restriction enzymes sensitive to adenine methylation.

[Two-stage revision procedure for septic complicated endoprosthesis of the hip]. / Dwuetapowa replantacja stawu biodrowego w leczeniu powiklan zapalnych endoplastyki.

Total hip arthroplasty is commonly used way for treatment of degenerative changes of a hip joint. Continuous progress of medicine enables use of more and more modern implants, that are supposed to provide to patients comfortable and painless motility. Nevertheless, with increasing number of implanted endoprostheses, rising numer of loosened impants is observed. Proper laboratory dignostics and postoperative proceedings as well as systematic follow up with standard X-ray scans allow for early recognition of loosened prostheses and implementation of surgical procedure. In the article two-stage method for treatment of septic loosened primary total hip arthroplasties applied in CMKP Orthopedic Clinic in Otwock, Poland as well as results of treatment of selected cases are presented.

Malignant neoplasm of cecum in Rendu-Osler-Weber syndrome patient.

The presented case is the another one which ilustates the coexistence of cecum malignancy and genetics-conditional multisystemic arteriovenous malformations. A 64-year-old male with the mucosal form of Rendu-Osler-Weber syndrome presented a history of two different causes of lower gastrointestinal bleeding occurred clinically and confirmed by colonoscopy. The more dangerous one there was the malignant process in the cecum. The right hemicolectomy was successfully performed for malignancy removal. The postoperative course was uneventful. Repeated gastrointestinal bleedings in the course of Rendu-Osler-Weber syndrome could readily hide the developmental malignant process within the colon. The main clinical implication is the mucosal form of Rendu-Osler-Weber syndrome not excludes any malignant transformation within the gastrointestinal tract. The another one is the necessity of colonoscopy and gastroscopy in every case of gastrointestinal bleeding event.

The activity of ornithine transcarbamoylase and arginase during mechanical jaundice in the rat model.

BACKGROUND: The pathogenesis of morbidity and mortality associated with intervention of the biliary system in patients with obstructive jaundice is unknown. Mechanical jaundice initiates the development of morphological changes in hepatocytes with concomitant disturbances in metabolism. These are followed by changes in enzyme activity in hepatocytes and peripheral blood. MATERIAL AND METHODS: Wistar rats were divided into three groups: group 1, sham-operated controls; group 2, rats with permanent jaundice; and group 3, rats with temporary mechanical jaundice. The animals were examined at 2 weeks (groups A), 4 weeks (groups B) and 6 weeks (groups C) after surgery. We explored the impact of induced mechanical jaundice on the activity of selected urea cycle enzymes (arginase [E.C.3.5.3.1] and ornithine transcarbamoylase (OTC) [E.C.2.1.3.3]). RESULTS: Mechanical jaundice was found to induce changes in hepatocytic metabolism, which in turn led to disturbances in the urea cycle and the process of transamination. After relief of the mechanical jaundice (recanalization of the common bile duct), the urea cycle activity in the liver was greatly increased despite the normalization of the basic biochemical indices. CONCLUSION: The results of the experiment confirmed the hypothesis that long-term mechanical jaundice causes lasting disturbances in hepatocytic metabolism. We conclude that the rate of nitrogen metabolism is higher after recanalization of the bile duct.