Nature-inspired peptide of MtDef4 C-terminus tail enables protein delivery in mammalian cells.

Cell-penetrating peptides show promise as versatile tools for intracellular delivery of therapeutic agents. Various peptides have originated from natural proteins with antimicrobial activity. We investigated the mammalian cell-penetrating properties of a 16-residue peptide with the sequence GRCRGFRRRCFCTTHC from the C-terminus tail of the Medicago truncatula defensin MtDef4. We evaluated the peptide's ability to penetrate multiple cell types. Our results demonstrate that the peptide efficiently penetrates mammalian cells within minutes and at a micromolar concentration. Moreover, upon N-terminal fusion to the fluorescent protein GFP, the peptide efficiently delivers GFP into the cells. Despite its remarkable cellular permeability, the peptide has only a minor effect on cellular viability, making it a promising candidate for developing a cell-penetrating peptide with potential therapeutic applications.

Kidney-specific methylation patterns correlate with kidney function and are lost upon kidney disease progression.

BACKGROUND: Chronological and biological age correlate with DNA methylation levels at specific sites in the genome. Linear combinations of multiple methylation sites, termed epigenetic clocks, can inform us the chronological age and predict multiple health-related outcomes. However, why some sites correlating with lifespan, healthspan, or specific medical conditions remain poorly understood. Kidney fibrosis is the common pathway for chronic kidney disease, which affects 10% of European and US populations. RESULTS: Here we identify epigenetic clocks and methylation sites that correlate with kidney function. Moreover, we identify methylation sites that have a unique methylation signature in the kidney. Methylation levels in majority of these sites correlate with kidney state and function. When kidney function deteriorates, all of these sites regress toward the common methylation pattern observed in other tissues. Interestingly, while the majority of sites are less methylated in the kidney and become more methylated with loss of function, a fraction of the sites are highly methylated in the kidney and become less methylated when kidney function declines. These methylation sites are enriched for specific transcription-factor binding sites. In a large subset of sites, changes in methylation patterns are accompanied by changes in gene expression in kidneys of chronic kidney disease patients. CONCLUSIONS: These results support the information theory of aging, and the hypothesis that the unique tissue identity, as captured by methylation patterns, is lost as tissue function declines. However, this information loss is not random, but guided toward a baseline that is dependent on the genomic loci. SIGNIFICANCE STATEMENT: DNA methylation at specific sites accurately reflects chronological and biological age. We identify sites that have a unique methylation pattern in the kidney. Methylation levels in the majority of these sites correlate with kidney state and function. Moreover, when kidney function deteriorates, all of these sites regress toward the common methylation pattern observed in other tissues. Thus, the unique methylation signature of the kidney is degraded, and epigenetic information is lost, when kidney disease progresses. These methylation sites are enriched for specific and methylation-sensitive transcription-factor binding sites, and associated genes show disease-dependent changes in expression. These results support the information theory of aging, and the hypothesis that the unique tissue identity, as captured by methylation patterns, is lost as tissue function declines.

Immunorecognition of Streptococcus mutans secreted proteins protects against caries by limiting tooth adhesion.

INTRODUCTION: Childhood caries, a prevalent chronic disease, affects 60-90 % of children in industrialized regions, leading to lesions in both primary and permanent teeth. This condition precipitates hospital admissions, emergency room visits, elevated treatment costs, and missed school days, thereby impeding the child's academic engagement and increasing the likelihood of caries into adulthood. Despite multiple identified risk factors, significant interpersonal variability remains unexplained. The immune system generates a unique antibody repertoire, essential for maintaining a balanced and healthy oral microbiome. Streptococcus mutans is a primary contributor to the development of caries. METHODS: Employing mass spectrometry, we investigated the S. mutans proteins targeted by antibodies in children both with and without caries, delineating a fundamental suite of proteins discernible by the immune systems of a majority of individuals. Notably, this suite was enriched with proteins pivotal for bacterial adhesion. To ascertain the physiological implications of these discoveries, we evaluated the efficacy of saliva in thwarting S. mutans adherence to dental surfaces. RESULTS: Antibodies in most children recognized a core set of ten S. mutans proteins, with additional proteins identified in some individuals. There was no significant difference in the proteins identified by children with or without caries, but there was variation in antibody binding intensity to some proteins. Functionally, saliva from caries-free individuals, but not children with caries, was found to hinder the binding of S. mutans to teeth. These findings delineate the S. mutans proteome targeted by the immune system and suggest that the inhibition of bacterial adherence to teeth is a primary mechanism employed by the immune system to maintain oral balance and prevent caries formation. CONCLUSIONS: These findings enhance our knowledge of the immune system's function in oral health maintenance and caries prevention, shedding light on how immunoglobulins interact with S. mutans proteins. CLINICAL SIGNIFICANCE: Targeting S. mutans proteins implicated in bacterial adhesion could be a promising strategy for preventing childhood caries.

A novel computationally engineered collagenase reduces the force required for tooth extraction in an ex-situ porcine jaw model.

The currently employed tooth extraction methods in dentistry involve mechanical disruption of the periodontal ligament fibers, leading to inevitable trauma to the bundle bone comprising the socket walls. In our previous work, we have shown that a recombinantly expressed truncated version of clostridial collagenase G (ColG) purified from Escherichia coli efficiently reduced the force needed for tooth extraction in an ex-situ porcine jaw model, when injected into the periodontal ligament. Considering that enhanced thermostability often leads to higher enzymatic activity and to set the basis for additional rounds of optimization, we used a computational protein design approach to generate an enzyme to be more thermostable while conserving the key catalytic residues. This process generated a novel collagenase (ColG-variant) harboring sixteen mutations compared to ColG, with a nearly 4℃ increase in melting temperature. Herein, we explored the potential of ColG-variant to further decrease the physical effort required for tooth delivery using our established ex-situ porcine jaw model. An average reduction of 11% was recorded in the force applied to extract roots of mandibular split first and second premolar teeth treated with ColG-variant, relative to those treated with ColG. Our results show for the first time the potential of engineering enzyme properties for dental medicine and further contribute to minimally invasive tooth extraction.

Enhancement of Collagen-I Levels in Human Gingival Fibroblasts by Small Molecule Activation of HIF-1α.

Collagen is the most abundant protein in various mammalian tissues and has an essential role in various cellular processes. Collagen is necessary for food-related biotechnological applications such as cultivated meat, medical engineering, and cosmetics. High-yield expression of natural collagen from mammalian cells is challenging and not cost-effective. Thus, external collagen is obtained primarily from animal tissues. Under cellular hypoxia, overactivation of the transcription factor hypoxia-inducible factor (HIF) was shown to correlate with enhanced accumulation of collagen. Herein, we showed that the small molecule ML228, a known molecular activator of HIF, enhances the accumulation of collagen type-I in human fibroblast cells. We report an increase in collagen levels by 2.33 ± 0.33 when fibroblasts were incubated with 5 µM of ML228. Our experimental results demonstrated, for the first time, that external modulation of the hypoxia biological pathway can boost collagen levels in mammalian cells. Our findings pave the way for enhancing natural collagen production in mammals by altering cellular signaling pathways.

Single-cell transcriptomic analysis of oral masticatory and lining mucosa-derived mesenchymal stromal cells.

AIM: To reveal the heterogeneity of ex vivo-cultured human mesenchymal stromal cells derived from either masticatory or lining oral mucosa. MATERIALS AND METHODS: Cells were retrieved from the lamina propria of the hard palate and alveolar mucosa of three individuals. The analysis of transcriptomic-level differences was accomplished using single-cell RNA sequencing. RESULTS: Cluster analysis clearly distinguished between cells from the masticatory and lining oral mucosa, and revealed 11 distinct cell sub-populations, annotated as fibroblasts, smooth muscle cells or mesenchymal stem cells. Interestingly, cells presenting a mesenchymal stem cell-like gene expression pattern were predominantly found in masticatory mucosa. Although cells of masticatory mucosa origin were highly enriched for biological processes associated with wound healing, those from the lining oral mucosa were highly enriched for biological processes associated with the regulation of epithelial cells. CONCLUSIONS: Our previous work had shown that cells from the lining and masticatory oral mucosae are phenotypically heterogeneous. Here, we extend these findings to show that these changes are not the result of differences in averages but rather represent two distinct cell populations, with mesenchymal stem cells more common in masticatory mucosa. These features may contribute to specific physiological functions and have relevance for potential therapeutic interventions.

The Hidden Secrets of the Dental Calculus: Calibration of a Mass Spectrometry Protocol for Dental Calculus Protein Analysis.

Dental calculus is a solid deposit that forms and accumulates on the tooth surface, entrapping oral microorganisms, biomolecules, and other micro-debris found in the oral cavity. A mass spectrometry analysis of its protein content opens a vista into the subject's diet, oral flora, and even some aspects of health, thus providing new insight and expanding our knowledge of archaic cultures. Multiple experimental protocols have been proposed for the optimal extraction of proteins from dental calculus. Herein, we compared various experimental conditions in order to calibrate and validate a protocol for protein extraction. Our results show that a high concentration of acetic acid followed by mechanical crushing and sonication provided the highest protein yield, while acetone precipitation enabled the identification of more distinct proteins. We validated this protocol using archeological samples, identifying human and microbial proteins in specimens from the eighth and seventeenth centuries (approximately 250-1300 years ago). These findings demonstrate that the developed protocol is useful for studying excavated archaeological samples and that it might be utilized to explore the biohistory, dietary habits, and microbiome of archaic populations.

The substrate scope of dehydratases in antibiotic biosynthesis and their application in kinetic resolutions.

Nine dehydratases involved in the biosynthesis of secondary metabolites in addition to FabZ from fatty acid biosynthesis were investigated for their substrate scope using a panel of N-acetylcysteamine (SNAC) thioesters. The best performing enzyme BorDH2 was applied in kinetic resolutions.

Origin of the 3-methylglutaryl moiety in caprazamycin biosynthesis.

BACKGROUND: Caprazamycins are liponucleoside antibiotics showing bioactivity against Gram-positive bacteria including clinically relevant Mycobacterium tuberculosis by targeting the bacterial MraY-translocase. Their chemical structure contains a unique 3-methylglutaryl moiety which they only share with the closely related liposidomycins. Although the biosynthesis of caprazamycin is understood to some extent, the origin of 3-methylglutaryl-CoA for caprazamycin biosynthesis remains elusive. RESULTS: In this work, we demonstrate two pathways of the heterologous producer Streptomyces coelicolor M1154 capable of supplying 3-methylglutaryl-CoA: One is encoded by the caprazamycin gene cluster itself including the 3-hydroxy-3-methylglutaryl-CoA synthase Cpz5. The second pathway is part of primary metabolism of the host cell and encodes for the leucine/isovalerate utilization pathway (Liu-pathway). We could identify the liu cluster in S. coelicolor M1154 and gene deletions showed that the intermediate 3-methylglutaconyl-CoA is used for 3-methylglutaryl-CoA biosynthesis. This is the first report of this intermediate being hijacked for secondary metabolite biosynthesis. Furthermore, Cpz20 and Cpz25 from the caprazamycin gene cluster were found to be part of a common route after both individual pathways are merged together. CONCLUSIONS: The unique 3-methylglutaryl moiety in caprazamycin originates both from the caprazamycin gene cluster and the leucine/isovalerate utilization pathway of the heterologous host. Our study enhanced the knowledge on the caprazamycin biosynthesis and points out the importance of primary metabolism of the host cell for biosynthesis of natural products.

Fibroblasts from the oral masticatory and lining mucosa have different gene expression profiles and proliferation rates.

AIMS: To compare the gene expression profiles and proliferation rates of fibroblasts from the oral lining and masticatory mucosae. MATERIALS AND METHODS: Primary human fibroblasts were retrieved from the posterior masticatory hard palate and the lining alveolar mucosa of five individuals. The gene expression profile was evaluated using total RNA sequencing. The proliferation rate was determined colorimetrically. RESULTS: Substantial differences in specific gene groups and pathways were observed between fibroblasts from the two tissues. Significantly enriched gene ontology processes were focused on the extracellular components. Lining mucosa fibroblasts exhibited significantly higher expression of the principal structural collagens, cranial neural crest markers, and homeobox genes associated with positional memory. Masticatory mucosa fibroblasts showed greater expression of genes related to transforming growth factor-ß signalling, which may be associated with fibrosis. In addition, they expressed higher levels of the EP2 prostaglandin E2 receptor and Toll-like receptor 1. Finally, masticatory mucosa fibroblasts exhibited a 10%-30% higher proliferation rate. CONCLUSIONS: Fibroblasts from the lining and masticatory oral mucosae are phenotypically heterogeneous, presenting distinct gene expression profiles and proliferation rates. These features may contribute to their specific physiological functions and have relevance for potential therapeutic applications.

Lamin regulates the dietary restriction response via the mTOR pathway in Caenorhabditis elegans.

Animals subjected to dietary restriction (DR) have reduced body size, low fecundity, slower development, lower fat content and longer life span. We identified lamin as a regulator of multiple dietary restriction phenotypes. Downregulation of lmn-1, the single Caenorhabditis elegans lamin gene, increased animal size and fat content specifically in DR animals. The LMN-1 protein acts in the mTOR pathway, upstream of RAPTOR and S6 kinase ß1 (S6K), a key component of and target of the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1), respectively. DR excludes the mTORC1 activator RAGC-1 from the nucleus. Downregulation of lmn-1 restores RAGC-1 to the nucleus, a necessary step for the activation of the mTOR pathway. These findings further link lamin to metabolic regulation.

Exploring the nuclear lamina in health and pathology using C. elegans.

The eukaryotic genome inside the nucleus is enveloped by two membranes, the Outer Nuclear Membrane (ONM) and the Inner Nuclear Membrane (INM). Tethered to the INM is the nuclear lamina, a fibrillar network composed of lamins-the nuclear intermediate filaments, and membrane associated proteins. The nuclear lamina interacts with several nuclear structures, including chromatin. As most nuclear functions, including regulation of gene expression, chromosome segregation and duplication as well as nuclear structure, are highly conserved in metazoans, the Caenorhabditis elegans nematode serves as a powerful model organism to study nuclear processes and architecture. This translucent organism can easily be observed under a microscope as a live embryo, larvae and even adult. Here we will review the data on nuclear lamina composition and functions gathered from studies using C. elegans model organisms: We will discuss genome spatial organization and its contribution to gene expression. We will review both the interaction between the cytoplasm and the nucleus and mechanotransduction mechanism. Finally, we will discuss disease causing mutation in nuclear lamins, including the use of this animal model in diseases research.

Rituximab biosimilar RTXM83 versus reference rituximab in combination with CHOP as first-line treatment for diffuse large B-cell lymphoma: a randomized, double-blind study.

This multicenter, double-blind, randomized study compared the efficacy, pharmacokinetics (PKs)/pharmacodynamics (PDs), safety and immunogenicity profile of RTXM83 vs. reference rituximab (R-rituximab), both with CHOP, as first-line treatment of diffuse large B-cell lymphoma (DLBCL). A total of 272 patients <65 years of age, with good prognosis (136 per arm) were randomized (1:1) to receive six cycles of either RTXM83 or R-rituximab. The primary efficacy endpoint was achieved (overall response rate of 83.6% for RTXM83 and 82.9% for R-rituximab) with a difference 0.7% between arms (95%CI: [-8.77% to 10.17%]) fulfilling the predefined non-inferiority margin (-13%). Similar number of patients reported at least one adverse event (AE) (131 per arm) or one serious AE (47 with RTXM83 and 45 with R-rituximab). Anti-drug antibody development was comparable between the arms. PK/PD secondary endpoint results support similarity between the compounds. RTXM83 exhibits non-inferior efficacy and similar safety/immunogenicity to R-rituximab, being an accessible alternative for the treatment of patients with previously untreated DLBCL.

Antibody-Driven Proximity Labeling in Fixed Tissues.

Protein function often depends on assemblies and interactions. These show complex spatial and temporal organization within the cell. Analysis of protein function can be greatly assisted by grouping proteins with their neighbors. Rather than relying on affinity, proximity labeling targets proteins proximal to the target of interest. We describe a protocol for antibody-guided deposition of tags in fixed and permeabilized cell lines and primary human tissue samples.

Small GTPases in C. elegans metabolism.

The mechanistic target of rapamycin (mTOR) is an evolutionary conserved protein with a serine/threonine kinase activity that regulates cell growth, proliferation, motility, survival, protein synthesis, autophagy and transcription. It is embedded in 2 large protein complexes: mTORC1 and mTORC2. Regulation of specific mTOR pathway functions depends on multiple GTPases, that act either as regulators of mTOR protein complexes, coupling energy availability with mTORC1 activity, or as downstream effectors of both mTORC1 and mTORC2. In this commentary, we highlight the advantages of studying the mTOR pathway in C. elegans, including the subcellular localization of the signaling pathway components and the animal phenotypes following tissue specific protein over-expression or knockdown. One important regulator that is not limited to the mTOR pathway is RHEB. We discuss in vitro and in vivo data suggesting that RHEB can function as an inhibitor of mTOR when not bound to GTP. RHEB-1 itself is regulated by Rab GDP dissociation inhibitor ß, which directly binds to ATX-2. We also highlight the roles of these proteins in dietary restriction-depended reduction in animal size and fat content.

Biotinylation by antibody recognition-a method for proximity labeling.

The high-throughput detection of organelle composition and proteomic mapping of protein environment directly from primary tissue as well as the identification of interactors of insoluble proteins that form higher-order structures have remained challenges in biological research. We report a proximity-based labeling approach that uses an antibody to a target antigen to guide biotin deposition onto adjacent proteins in fixed cells and primary tissues, which allows proteins in close proximity to the target antigen to be captured and identified by mass spectrometry. We demonstrated the specificity and sensitivity of our method by examining the well-studied mitochondrial matrix. We then used the method to profile the dynamic interactome of lamin A/C in multiple cell and tissue types under various treatment conditions. The ability to detect proximal proteins and putative interactors in intact tissues, and to quantify changes caused by different conditions or in the presence of disease mutations, can provide a window into cell biology and disease pathogenesis.

A novel somatic mutation achieves partial rescue in a child with Hutchinson-Gilford progeria syndrome.

BACKGROUND: Hutchinson-Gilford progeria syndrome (HGPS) is a fatal sporadic autosomal dominant premature ageing disease caused by single base mutations that optimise a cryptic splice site within exon 11 of the LMNA gene. The resultant disease-causing protein, progerin, acts as a dominant negative. Disease severity relies partly on progerin levels. METHODS AND RESULTS: We report a novel form of somatic mosaicism, where a child possessed two cell populations with different HGPS disease-producing mutations of the same nucleotide-one producing severe HGPS and one mild HGPS. The proband possessed an intermediate phenotype. The mosaicism was initially discovered when Sanger sequencing showed a c.1968+2T>A mutation in blood DNA and a c.1968+2T>C in DNA from cultured fibroblasts. Deep sequencing of DNA from the proband's blood revealed 4.7% c.1968+2T>C mutation, and 41.3% c.1968+2T>A mutation. CONCLUSIONS: We hypothesise that the germline mutation was c.1968+2T>A, but a rescue event occurred during early development, where the somatic mutation from A to C at 1968+2 provided a selective advantage. This type of mosaicism where a partial phenotypic rescue event results from a second but milder disease-causing mutation in the same nucleotide has not been previously characterised for any disease.

The effect of metabolic comorbidities and commonly used drugs on the prognosis of patients with ovarian cancer.

BACKGROUND: Diabetes mellitus is associated with an increased risk of recurrence in patients with ovarian cancer. Retrospective studies suggested that the use of statins, metformin and beta blockers is associated with improved prognosis in these patients. Patients with diabetes often suffer from hypertension and are usually treated concomitantly by several classes of drugs. Our aim was to assess the independent contribution of diabetes mellitus and hypertension and of the use of aspirin, statins, metformin and beta blockers on the risk of ovarian cancer recurrence and mortality. METHODS: Files of ovarian cancer patients treated between 2000 and 2012 were retrospectively reviewed. Data regarding disease characteristics, presence of diabetes mellitus and hypertension, recurrence and death were extracted. The use of drugs was assessed using the Clalit Health Services (CHS) pharmacy records. RESULTS: 143 patients treated by debulking surgery and platinum based chemotherapy were included. Median age was 62.5, 22 (15.4%) had diabetes mellitus, 61 (42.7%) had chronic hypertension. Statins were used by 43 (30%) patients, 31 (21.7%) used aspirin, 25 (17.5%) used beta blockers and 12 (8.4%) used metformin. In multivariate analysis diabetes mellitus was associated with a shorter recurrence free survival (RFS) and the use of aspirin and metformin was associated with a prolonged RFS in this cohort. Overall survival (OS) was longer in patients using aspirin and shorter in patients with hypertension. CONCLUSIONS: Our data suggests that metabolic comorbidities and commonly used drugs are associated with the prognosis of patients with ovarian cancer. Additional trials are needed to confirm these observations and test therapeutic interventions.

Vibration-induced electrical noise in a cryogen-free dilution refrigerator: Characterization, mitigation, and impact on qubit coherence.

Cryogen-free low-temperature setups are becoming more prominent in experimental science due to their convenience and reliability, and concern about the increasing scarcity of helium as a natural resource. Despite not having any moving parts at the cold end, pulse tube cryocoolers introduce vibrations that can be detrimental to the experiments. We characterize the coupling of these vibrations to the electrical signal observed on cables installed in a cryogen-free dilution refrigerator. The dominant electrical noise is in the 5-10 kHz range and its magnitude is found to be strongly temperature dependent. We test the performance of different cables designed to diagnose and tackle the noise, and find triboelectrics to be the dominant mechanism coupling the vibrations to the electrical signal. Flattening a semi-rigid cable or jacketing a flexible cable in order to restrict movement within the cable, successfully reduces the noise level by over an order of magnitude. Furthermore, we characterize the effect of the pulse tube vibrations on an electron spin qubit device in this setup. Coherence measurements are used to map out the spectrum of the noise experienced by the qubit, revealing spectral components matching the spectral signature of the pulse tube.