Endogenous Marker Research Articles

CYSTATIN C AS A MARKER OF EARLY KIDNEY DAMAGE UNDER ARTERIAL HYPERTENSION (LITERATURE REVIEW)

The purpose of this study is to determine kidney functional activity in relation to cystatin C levels, as reported in recent literature. Materials and Methods. A bibliographic-semantic method was employed to assess the current state of research on this topic, analyzing findings from previous scientific studies using both literature sources and electronic resources. Results. Chronic kidney disease (CKD) is as prevalent as hypertension. It is well-known that arterial hypertension exacerbates kidney function, making the assessment of renal functional activity critical. Glomerular filtration rate (GFR) is commonly used to evaluate kidney function, relying on established indicators as well as newer, more objective markers. Among these, cystatin C has emerged as one of the most accurate and sensitive indicators for assessing kidney function. Its concentration in serum negatively correlates with GFR and is particularly valuable for detecting renal pathology even when creatinine levels remain unchanged. This insight has led to the development of an estimated GFR (eGFR) formula that incorporates cystatin C levels. Research suggests that cystatin C levels increase with hypertension, although studies on the specific relationship between cystatin C and hypertension remain limited. Some findings propose that serum cystatin C could serve as a predictor of disease severity, particularly in elderly hypertensive patients with coronary heart disease. The literature supports the utility of cystatin C as a reliable marker for assessing GFR, which facilitates early detection of CKD even when albumin excretion is normal, thus identifying kidney damage at an early stage. Ppotential mechanisms by which elevated cystatin C may contribute to cardiovascular damage are under investigation. As a highly informative endogenous marker of GFR, serum cystatin C not only aids in staging CKD but is especially valuable in identifying early renal dysfunction. Conclusion. The measurement of cystatin C enables a highly accurate assessment of kidney function and aids in evaluating cardiovascular risk, especially when hypertension coexists with chronic kidney disease. Further research is needed to improve predictions of chronic kidney progression at various stages and in the presence of comorbidities. Such insights will support more effective preventive strategies to slow the progression of this pathology.

Identification of the proliferative activity of germline progenitor cells in the adult ovary of the bat Artibeus jamaicensis.

Until a few years ago, it was assumed that oocyte renewal did not take place in the ovary of adult organisms; however, the existence of germline progenitor cells (GPCs), which renew the ovarian follicular reserve, has now been documented in mammals. Specifically, in the adult ovary of bats, the presence of cells located in the cortical region with characteristics similar to GPCs, called adult cortical germ cells (ACGC), has been observed. One of the requirements that a GPC must fulfil is to be able to proliferate mitotically, so the evaluation of cell proliferation in ACGC is of utmost importance in order to be able to relate them to a parental lineage. Currently, there are several methods to determine cell proliferation, including BrdU labelling or the use of endogenous proliferation markers. Thus, the aim of this work was to evaluate the proliferative activity of ACGC in the adult ovary of the bat Artibeus jamaicensis, using different proliferation markers and correlating these with the protein expression of the transcription factor Oct4 and the germ line marker Ddx4. We found that the expression pattern of the proliferation markers BrdU, PCNA, Ki-67 and pH3 occurs at different times of the cell cycle, so co-localization of two or more of these markers allows us to identify proliferating cells. This allowed us to identify ACGC with proliferative capacity in the adult ovary of A. jamaicensis, suggesting that GPCs renew the follicle reserve during the adult life of the organism.

Evaluating the nutrient composition and antioxidant properties of orange (Citrus sinensis) peels through Penicillium camemberti-based solid-substrate fermentation

Citrus by-products, especially orange peels, are a significant global waste issue, driving research into management solutions. Solid-substrate fermentation is a promising method to convert this waste into valuable resources, enhancing its medicinal and nutritional potential while addressing environmental and economic concerns in food waste management. Therefore, this study was carried out to evaluate the impact of solid-substrate fermentation (SSF) with Penicillium camemberti on the nutrient profile, antioxidative properties, and potential neuroprotective effects of orange peels in AlCl3 (Al)-induced neurotoxicity in fruit fly (Drosophila melanogaster) model. Orange peels were collected, air-dried to a constant weight, and subjected to a 10-day SSF process with Penicillium camemberti. Post-fermentation, in vitro nutritional profiling, and antioxidant assays were conducted, alongside in vivo assessments of the neuroprotective effects on survival rate, memory index, monoamine oxidase (MAO) activity, and endogenous antioxidant markers on Al-induced fruit flies were determined. The results showed that fermentation with Penicillium camemberti significantly enhanced the nutritional composition of the orange peels compared to their unfermented counterparts. The antioxidant properties of fermented orange peels were significantly (p < 0.05) increased, as evidenced by increased DPPH scavenging activity (81.68 ± 0.18) and ferric reducing antioxidant power (10.77 ± 0.27). Additionally, biochemical assays in vivo revealed a significant improvement in survival rates and memory indices in Al-induced flies. Also, monoamine oxidase (MAO) levels were significantly reduced, while glutathione S-transferase (GST) levels were significantly (p < 0.05) elevated in the induced flies fed with the fermented samples. Conclusively, this study highlights the capability of Penicillium camemberti fermentation in converting waste into valuable resources, enhancing the nutrient profile of citrus peels for applications in livestock feed and potential therapeutic interventions for degenerative disorders.

The bronchoalveolar lavage dilution conundrum: an updated view on a long-standing problem.

Bronchoalveolar lavage (BAL) is used by researchers to study molecular interactions within healthy and diseased human lungs. However, the utility of BAL fluid measurements may be limited by difficulties accounting for dilution of the epithelial lining fluid (ELF) sampled and inconsistent collection techniques. The use of endogenous markers to estimate ELF dilution has been proposed as a potential method to normalize acellular molecule measurements in BAL fluid, but these markers are also imperfect and prone to inaccuracy. The focus of this report is to review factors that affect the interpretation of acellular molecule measurements in lung ELF and present original data comparing the performance of several BAL dilution markers during health and in a human endobronchial endotoxin challenge model of acute inflammation. Our findings suggest that incomplete ELF and lavage fluid mixing, flux of markers across the alveolar barrier, and lung inflammation are all possible factors that can affect marker performance. Accounting for these factors, we show that commonly used markers including urea, total protein, albumin, and immunoglobulin M are likely unreliable BAL dilution markers. In contrast, surfactant protein D appears to be less affected by these factors and may be a more accurate and biologically plausible marker to improve the reproducibility of acellular BAL component measurements across individuals during health and inflammatory states.NEW & NOTEWORTHY In this report, mathematical prediction models and real-world measurements are used to compare the performance of molecular markers of dilution in bronchoalveolar lavage fluid samples. Effects of acute inflammation within individual subjects are highlighted. These findings inform recommendations for normalizing measurements across bronchoalveolar lavage samples and highlight the need for additional markers to improve the rigor of translational studies utilizing bronchoalveolar lavage measurements.

9194 Generation of a Human-Induced Pluripotent Stem Cell Line From a Patient With Hypopituitarism and a Novel Nonsense Variant in FOXA2

Abstract Disclosure: M.A. Camilletti: None. G.T. Chirino Felker: None. G. Amin: None. S. Castañeda: None. F. Zabalegui: None. C. Romano Florit: None. A. Waisman: None. M. Ciaccio: None. M.I. Di Palma: None. E. Vaiani: None. A. Belgorosky: None. S.G. Miriuka: None. L.N. Moro: None. M.I. Perez-Millan: None. Congenital Hypopituitarism (CH) is a genetically complex disease characterized by one or more pituitary hormone deficiencies. Forkhead Box A2 (FOXA2, 600288) is a pioneer transcription factor associated with development of multiple tissues in humans and mice. Heterozygous loss of function mutations in FOXA2 were recently shown to cause CH with hyperinsulinemic hypoglycemia, but little is known about the molecular mechanism of FOXA2 action during pituitary development. We identified a novel, heterozygous nonsense variant in FOXA2 (c.686C&amp;lt;A; p.S229X) in a patient diagnosed with GH deficiency at 1.7 years of age, with anterior lobe hypoplasia and absent pituitary stalk, asymptomatic hypoglycemia, and craniofacial malformations. To discover the mechanism of FOXA2 regulation of pituitary development we generated an induced pluripotent stem cell line (FOXA2mut-iPSC) from the patient’s polymorphonuclear blood cells (PBMCs) using an EF1a-hSTEMCCA-loxP vector containing the pluripotency genes OCT-4, SOX-2, c-MYC and KLF4. FOXA2mut-iPSC clone R1 had a normal karyotype and embryonic stem cell-like morphology. These cells express endogenous pluripotency-associated markers NANOG, SOX2, and OCT4, have alkaline phosphatase activity, and can differentiate into all three developmental layers, indicating that these cells are pluripotent. Short tandem repeat (STR) analysis confirmed that FOXA2mut-iPSC R1 clone profiles matched those of the donor PBMCs. Next, we compared pituitary differentiation of FOXA2mut-iPSC vs. control-iPSCs in vitro. Cells were cultured in a differentiation medium containing BMP4, the smoothened agonist SAG, and fibroblast growth factor (FGF), as described in Zimmer et al., 2016 (1). Gene expression analysis (by qRT-PCR) of samples collected on days 0, 4, 7, and 15 of the differentiation protocol showed an increased expression of genes associated with anterior pituitary development including OTX2, PITX1/2, and SIX1 in both cell lines (n=2). These preliminary data suggest that pituitary cell fate can be induced in the heterozygous FOXA2mut-iPSC clone. Ongoing studies will assess pituitary hormone-producing cell differentiation after longer periods of FOXA2mut-iPSC cell culture. In sum, patient iPSC differentiation is a promising tool for assessment of normal and variant FOXA2 function in human pituitary development, and for enlightening the mechanisms of FOXA2 action in human pituitary development. (1) Zimmer et al., Stem Cell Reports. 2016 Jun 14;6(6):858-872. Presentation: 6/1/2024

7151 Generation of a Human-Induced Pluripotent Stem Cell Line From a Patient With Hypopituitarism and a Novel Nonsense Variant in FOXA2

Abstract Disclosure: M.A. Camilletti: None. G.T. Chirino Felker: None. G. Amin: None. S. Castañeda: None. F. Zabalegui: None. C. Romano Florit: None. A. Waisman: None. M. Ciaccio: None. M.I. Di Palma: None. E. Vaiani: None. A. Belgorosky: None. S.G. Miriuka: None. L.N. Moro: None. M.I. Perez-Millan: None. Congenital Hypopituitarism (CH) is a genetically complex disease characterized by one or more pituitary hormone deficiencies. Forkhead Box A2 (FOXA2, 600288) is a pioneer transcription factor associated with development of multiple tissues in humans and mice. Heterozygous loss of function mutations in FOXA2 were recently shown to cause CH with hyperinsulinemic hypoglycemia, but little is known about the molecular mechanism of FOXA2 action during pituitary development. We identified a novel, heterozygous nonsense variant in FOXA2 (c.686C&amp;lt;A; p.S229X) in a patient diagnosed with GH deficiency at 1.7 years of age, with anterior lobe hypoplasia and absent pituitary stalk, asymptomatic hypoglycemia, and craniofacial malformations. To discover the mechanism of FOXA2 regulation of pituitary development we generated an induced pluripotent stem cell line (FOXA2mut-iPSC) from the patient’s polymorphonuclear blood cells (PBMCs) using an EF1a-hSTEMCCA-loxP vector containing the pluripotency genes OCT-4, SOX-2, c-MYC and KLF4. FOXA2mut-iPSC clone R1 had a normal karyotype and embryonic stem cell-like morphology. These cells express endogenous pluripotency-associated markers NANOG, SOX2, and OCT4, have alkaline phosphatase activity, and can differentiate into all three developmental layers, indicating that these cells are pluripotent. Short tandem repeat (STR) analysis confirmed that FOXA2mut-iPSC R1 clone profiles matched those of the donor PBMCs. Next, we compared pituitary differentiation of FOXA2mut-iPSC vs. control-iPSCs in vitro. Cells were cultured in a differentiation medium containing BMP4, the smoothened agonist SAG, and fibroblast growth factor (FGF), as described in Zimmer et al., 2016 (1). Gene expression analysis (by qRT-PCR) of samples collected on days 0, 4, 7, and 15 of the differentiation protocol showed an increased expression of genes associated with anterior pituitary development including OTX2, PITX1/2, and SIX1 in both cell lines (n=2). These preliminary data suggest that pituitary cell fate can be induced in the heterozygous FOXA2mut-iPSC clone. Ongoing studies will assess pituitary hormone-producing cell differentiation after longer periods of FOXA2mut-iPSC cell culture. In sum, patient iPSC differentiation is a promising tool for assessment of normal and variant FOXA2 function in human pituitary development, and for enlightening the mechanisms of FOXA2 action in human pituitary development. (1) Zimmer et al., Stem Cell Reports. 2016 Jun 14;6(6):858-872. Presentation: 6/1/2024

Absolute quantitation of human serum cystatin C: candidate reference method by 15N-labeled recombinant protein isotope dilution UPLC-MS/MS.

Serum cystatin C (CysC) is a reliable and ideal endogenous marker for accurately assessing early changes in glomerular filtration rate (GFR), surpassing the limitations of creatinine-based estimated GFR. To improve the precision of GFR calculation, the development of strategies for accurately measuring serum CysC is crucial. In this study, the full-length CysC pure product and fully recombinant 15N-labeled CysC internal standard were subjected to protein cleavage. Subsequently, an LC-MS/MS method was developed for the absolute quantification ofserum CysC. The traceability of the method was assigned calibrator using the amino acid reference measurement procedure (RMP). It involved calibrating the instrument using an amino acid reference material with known amino acid concentrations for calibration and comparison purposes. The total imprecision of the method was determined to be≤8.2 %, and a lower functional limit of quantification (LLoQ) was achieved. The recoveries ranged from 97.36 to 103.26 %. The relative bias between this candidate RMP for measurement of ERM-DA471-IFCC and the target value was 1.74 %. The linearity response was observed within the concentration range of 0.21-10.13 mg/L, with a high R2 value of 0.999. The results obtained using our method was consistent with those obtained using other certified RMPs. With the establishment of this highly selective and accurate serum CysC measurement method, it is now possible to assess the correlation between immunoassay results of serum CysC and the intended target when discrepancies are suspected in the clinical setting.

Regional use of extracellular microvesicles of mesenchymal stromal cells in acute necrotizing pancreatitis in an experiment

Background. The significance of the problem of treatment of acute pancreatitis is due to an increase in the incidence with an increase in the number of necrotizing forms, accompanied by a high incidence of severe complications and high mortality.Objective. To identify the impact of regionally used extracellular microvesicles of mesenchymal stromal cells on the endogenous intoxication markers in acute necrotizing pancreatitis in the experiment.Material and methods. Acute pancreatitis was induced by the introduction of a 0.3 ml of 5% solution of non-ionic polyethylene glycol octylphenol ether detergent into the caudal part of the rat pancreas. The study was conducted on 42 adult Wistar rats, which were randomly divided into 4 groups. Group I (n=6) included intact animals, Group II (control group) (n=12) included rats with pancreatitis without treatment, Group III (n=12) consisted of rats with pancreatitis treated with analgesia + infusions of 0.9% sodium chloride solution (saline), Group IV (n=12) included rats with pancreatitis treated with analgesia+ saline infusions + regional application of extracellular microvesicles of mesenchymal stromal cells. Cells were obtained from the bone marrow of healthy animals. Microvesicles were obtained by differential centrifugation under sterile conditions. Microvesicles were administered one day after the pancreatitis induction through the catheter installed into the pathologically altered part of the pancreas. The dose of microvesicles was calculated as equivalent to (derived from) 1 million mesenchymal stromal cells. The hematological parameters, markers of the systemic manifestation of the pathological process (alpha-amylase, aspartate aminotransferase, alanine aminotransferase), the endogenous intoxication markers (lipid peroxidation activity, nitric oxide level), the systemic inflammatory response markers (tumor necrosis factor-alpha, interleukin-6) were studied on the 3rd and 7th day from the start of disease modeling.Results. Regional use of extracellular microvesicles of mesenchymal stromal cells in the treatment of acute experimental necrotizing pancreatitis at an early stage helped to normalize the level of blood platelets, reduce enzymeemia, elements of endogenous intoxication (interleukin-6, tumor necrosis factor-alpha), and the nitric oxide level.Conclusion. The early application of extracellular microvesicles of mesenchymal stromal cells in the treatment of acute necrotizing pancreatitis in an experiment has a positive effect on parameters, which are key links of pathogenesis and leading markers of this disease severity.

Doublecortin-immunoreactive neurons in the piriform cortex are sensitive to the long lasting effects of early life stress.

The olfactory system is a niche of continuous structural plasticity, holding postnatal proliferative neurogenesis in the olfactory bulbs and a population of immature neurons in the piriform cortex. These neurons in the piriform cortex are generated during embryonic development, retain the expression of immaturity markers such as doublecortin, and slowly mature and integrate into the olfactory circuit as the animal ages. To study how early life experiences affect this population of cortical immature neurons, we submitted mice of the C57/Bl6J strain to a protocol of maternal separation for 3 h per day from postnatal day 3 to postnatal day 21. Control mice were continuously with their mothers. After weaning, mice were undisturbed until 6 weeks of age, when they were weighted and tested in the elevated plus-maze, a standard test for anxiety-like behavior, to check for phenotypical effects. Mice were then perfused, and their brains processed for the immunofluorescent detection of doublecortin and the endogenous proliferation marker Ki67. We found that maternal separation induced a significant increase in the body weight of males, but not females. Further, maternally separated mice displayed increased exploratory-like behavior (i.e., head dipping, velocity and total distance traveled in the elevated plus maze), but no significant differences in anxiety-like behavior or corticosterone levels after behavioral testing. Finally, we observed a significant increase in the number of complex doublecortin neurons in the piriform cortex, but not in the olfactory bulbs, of mice submitted to maternal separation. Interestingly, most doublecortin neurons in the piriform cortex, but not the olfactory bulb, express the epigenetic reader MeCP2. In summary, mild early life stress results, during adolescence, in a male-specific increase in body weight, alteration of the exploratory behaviors, and an increase in doublecortin neurons in the piriform cortex, suggesting an alteration in their maturation process.

Differences in the Expression of Autophagy Markers Microtubule-Associated Protein Light Chains 3A and 3B in Oral Premalignant Lesions and Oral Squamous Cell Carcinoma: A Cross-Sectional Study.

Background Microtubule-associated protein light chains (LC) 3A and 3B are the structural proteins of the autophagosomal membrane widely used as endogenous autophagy markers. LC3A and LC3B autophagosomes reportedly have a distinct subcellular localization yet their role in the transition from premalignant to malignant phase remains unclear. This exploratory study aimed to investigate the expression of autophagy-related proteins LC3A and LC3B in oral premalignant lesions (OPL) and oral squamous cell carcinoma (OSCC) cases. Methodology A cross-sectional study was conducted on 100 OPL and 39 OSCC samples. OPL samples comprised both dysplastic and non-dysplastic lesions. The expression of LC3A and LC3B markers was evaluated in the study samples using immunohistochemistry and associated with dysplasia in OPL and with invasive OSCC versus OPL. Fisher's exact test was used for statistical analysis. Results There was a higher ratio of LC3A positivity in non-dysplastic OPL (31/38) compared to dysplastic premalignant lesions (36/62, p=0.017). There was a higher ratio of LC3B positivity in dysplastic OPL (16/62) compared to non-dysplastic lesions (4/38) with a trend towards statistical significance (p=0.075). There was no statistical difference in the ratio ofLC3A positivity between OSCC (23/39) and premalignant (67/100) lesions, while the ratio of LC3B marker positivity was higher in OSCC cases (18/39) relative to premalignant lesions (20/100, p=0.003). Conclusion Autophagy-related proteins LC3A and LC3B may have different roles to play in a disease context manner. LC3A is likely to be negatively associated with dysplasia in OPL while LC3B expression is positively associated with carcinogenesis of OSCC, possibly including dysplasia.

Multistep allelic conversion in mouse pre-implantation embryos by AAV vectors

Site-specific recombinases (SSRs) are critical for achieving precise spatiotemporal control of engineered alleles. These enzymes play a key role in facilitating the deletion or inversion of loci flanked by recombination sites, resulting in the activation or repression of endogenous genes, selection markers or reporter elements. However, multiple recombination in complex alleles can be laborious. To address this, a new and efficient method using AAV vectors has been developed to simplify the conversion of systems based on Cre, FLP, Dre and Vika recombinases. In this study, we present an effective method for ex vivo allele conversion using Cre, FLP (flippase), Dre, and Vika recombinases, employing adeno-associated viruses (AAV) as delivery vectors. AAVs enable efficient allele conversion with minimal toxicity in a reporter mouse line. Moreover, AAVs facilitate sequential allele conversion, essential for fully converting alleles with multiple recombination sites, typically found in conditional knockout mouse models. While simple allele conversions show a 100% efficiency rate, complex multiple conversions consistently achieve an 80% conversion rate. Overall, this strategy markedly reduces the need for animals and significantly speeds up the process of allele conversion, representing a significant improvement in genome engineering techniques.

Abstract We010: In Vivo Partial Reprogramming of Cardiomyocytes to a Molecularly Rejuvenated State Ameliorates Cardiac Failure

Introduction: i n vivo partial cell reprogramming through transient overexpression of Oct3/4 , Klf4 , Sox2 and cMyc (OKSM) transcription factors generates de-differentiated or molecularly rejuvenated cells that may contribute to tissue regeneration. However, not all cell types are equally amenable to reprogramming, and tools to induce cell type-specific reprogramming in vivo are scarce. Here, we generated cardiomyocyte-specific, “reprogrammable” mice (CM-OKSM) that enabled inducible OKSM expression specifically in this cell type, characterized by its remarkably poor regenerative capacity after birth. Using this model, we investigated if partial, in situ , cardiomyocyte reprogramming was feasible and able to enhance cardiac regeneration. Methods: To generate lineage-traceable CM-OKSM mice, we combined Cre recombination driven by a cardiomyocyte-specific promoter ( Myh6 ), conditional GFP labelling and conditional, doxycycline (dox)-inducible, control of OKSM expression. We administered 1 mg/ml dox in the drinking water of CM-OKSM mice, and investigated reprogramming at the molecular level (immunostaining, RT-qPCR, RNAseq, DNA methylation), histological level (H&amp;E, electron microscopy) and functional level (teratoma assay, echocardiography). We compared dox administration for six cycles of a two-day ON/five-day OFF regime, and for eighteen continuous days, to investigate partial and full reprogramming to pluripotency, respectively. We conducted studies in young adult mice (8 to 12 weeks old) and on older mice (9 months old) with heart failure, to assess the effects of cardiomyocyte reprogramming in cardiac performance. Results and Discussion: In young CM-OKSM mice, adult cardiomyocytes can be fully reprogrammed to pluripotency in vivo by eighteen-day overexpression of OKSM. This was evidenced by significant upregulation of endogenous pluripotency markers, loss of sarcomere organization, cell de-differentiation, and the appearance of GFP+ teratomas with trilineage contribution. We then found that cyclic OKSM induction prevented reacquisition of pluripotency and tumorigenesis, both in young and old mice. In the latter, partial reprogramming of cardiomyocytes induced significant rejuvenation of their epigenetic age and halted the progression of congestive heart failure. Our results confirm that the OKSM cocktail can reprogram adult mouse cardiomyocytes in vivo and that partial reprogramming can contribute to functional improvements in cardiac disease.

In Situ Synthesis and Assembly of Functional Materials and Devices in Living Systems.

ConspectusIntegrating functional materials and devices with living systems enables novel methods for recording, manipulating, or augmenting organisms not accessible by traditional chemical, optical, or genetic approaches. (The term "device" refers to the fundamental components of complex electronic systems, such as transistors, capacitors, conductors, and electrodes.) Typically, these advanced materials and devices are synthesized, either through chemical or physical reactions, outside the biological systems (ex situ) before they are integrated. This is due in part to the more limited repertoire of biocompatible chemical transformations available for assembling functional materials in vivo. Given that most of the assembled bulk materials are impermeable to cell membranes and cannot go through the blood-brain barrier (BBB), the external synthesis poses challenges when trying to interface these materials and devices with cells precisely and in a timely manner and at the micro- and nanoscale─a crucial requirement for modulating cellular functions. In contrast to presynthesis in a separate location, in situ assembly, wherein small molecules or building blocks are directly assembled into functional materials within a biological system at the desired site of action, has offered a potential solution for spatiotemporal and genetic control of material synthesis and assembly.In this Account, we highlight recent advances in spatially and temporally targeted functional material synthesis and assembly in living cells, tissues and animals and provide perspective on how they may enable novel probing, modulation, or augmentation of fundamental biology. We discuss several strategies, starting from the traditional nontargeted methods to targeted assembly of functional materials and devices based on the endogenous markers of the biological system. We then focus on genetically targeted assembly of functional materials, which employs enzymatic catalysis centers expressed in living systems to assemble functional materials in specific molecular-defined cell types. We introduce the recent efforts of our group to modulate membrane capacitance and neuron excitability using in situ synthesized electrically functional polymers in a genetically targetable manner. These advances demonstrate the promise of in situ synthesis and assembly of functional materials and devices, including the optogenetic polymerization developed by our lab, to interface with cells in a cellular- or subcellular-specific manner by incorporating genetic and/or optical control over material assembly. Finally, we discuss remaining challenges, areas for improvement, potential applications to other biological systems, and novel methods for the in situ synthesis of functional materials that could be elevated by incorporating genetic or material design strategies. As researchers expand the toolkit of biocompatible in situ functional material synthetic techniques, we anticipate that these advancements could potentially offer valuable tools for exploring biological systems and developing therapeutic solutions.

The Pseudo-Natural Product Tafbromin Selectively Targets the TAF1 Bromodomain 2.

Phenotypic assays detect small-molecule bioactivity at functionally relevant cellular sites, and inherently cover a variety of targets and mechanisms of action. They can uncover new small molecule-target pairs and may give rise to novel biological insights. By means of an osteoblast differentiation assay which employs a Hedgehog (Hh) signaling agonist as stimulus and which monitors an endogenous marker for osteoblasts, we identified a pyrrolo[3,4-g]quinoline (PQ) pseudo-natural product (PNP) class of osteogenesis inhibitors. The most potent PQ, termed Tafbromin, impairs canonical Hh signaling and modulates osteoblast differentiation through binding to the bromodomain 2 of the TATA-box binding protein-associated factor 1 (TAF1). Tafbromin is the most selective TAF1 bromodomain 2 ligand and promises to be an invaluable tool for the study of biological processes mediated by TAF1(2) bromodomains.

The Pseudo‐Natural Product Tafbromin Selectively Targets the TAF1 Bromodomain 2

AbstractPhenotypic assays detect small‐molecule bioactivity at functionally relevant cellular sites, and inherently cover a variety of targets and mechanisms of action. They can uncover new small molecule‐target pairs and may give rise to novel biological insights. By means of an osteoblast differentiation assay which employs a Hedgehog (Hh) signaling agonist as stimulus and which monitors an endogenous marker for osteoblasts, we identified a pyrrolo[3,4‐g]quinoline (PQ) pseudo‐natural product (PNP) class of osteogenesis inhibitors. The most potent PQ, termed Tafbromin, impairs canonical Hh signaling and modulates osteoblast differentiation through binding to the bromodomain 2 of the TATA‐box binding protein‐associated factor 1 (TAF1). Tafbromin is the most selective TAF1 bromodomain 2 ligand and promises to be an invaluable tool for the study of biological processes mediated by TAF1(2) bromodomains.

Protective effect of Phlogacanthus thyrsiflorus Nees against experimentally induced gastric mucosal lesions in rats: Experimental evidence from biochemical and histological analysis

Phlogacanthus thyrsiflorus Nees (Acanthaceae) is traditionally used in the North East Himalayan region to treat stomach issues, including gastritis. This study aimed to investigate the gastroprotective effect of 70% aqueous ethanol extract of Phlogacanthus thyrsiflorus flowers (PTE), standardized by HPTLC and LC-MS/MS, and to establish its possible mechanisms. The gastroprotective effect of PTE, at doses of 200 and 400 mg/kg body weight, was evaluated using both physical (pylorus ligation, PL; cold restraint stress, CRS) and chemical (ethanol, EtOH) ulcerogens-induced gastric ulcers in Wistar rats. Animals were randomly divided into control, ulcer control, positive control and PTE-treated groups, with PTE administered orally twice daily for 5 days. The protective effect of PTE was assessed through various gastric ulcer parameters, including gastric pH, gastric wall mucus, non-protein sulfhydryls (NP-SH) content, microvascular permeability, endogenous antioxidant markers, and gastric histopathology. HPTLC and LC-MS/MS confirmed the presence of gallic acid, naringenin, β-sitosterol and ascorbic acid in PTE. The active components of PTE showed significant dose-dependent inhibition of ulcer index, with reductions of 28.59-50.03% in PL, 27.69-53.23% in EtOH and 31.14-57.53% in CRS models (P< 0.01). Additionally, PTE at 400 mg/kg significantly increased gastric pH by 23.41%, mucus content by 33.69%, and NP-SH levels by 37.24%. PTE also demonstrated significant antioxidant potential and histopathological studies confirmed its protective effect. The study suggests that the anti-ulcer effect of PTE may be attributed to its ability to preserve adhered gastric mucus, exhibit antisecretory effects, and scavenge free radicals.

Glomerular Filtration Rate in Sick Neonates: A Focus on Cystatin C

Background: The use of creatinine as an endogenous marker of estimated glomerular filtration rate (eGFR) is well-established in clinical practice, despite its limitations. As a step toward limiting this drawback, cystatin C and various biomarkers came into use in determining renal function. In sick neonates, various pathologies may impair renal function thus, underscoring the need for accurate estimation of glomerular filtration rate (GFR). Aims and Objectives: This study aimed at evaluating the use of cystatin C in estimating the GFR of sick neonates in comparison to creatinine. Materials and Methods: A hospital-based descriptive study was conducted at a tertiary center in North Central, Nigeria, for 4 months. One hundred and seventy-three sick neonates admitted into the special care baby unit were recruited. Blood was sampled at admission for determination of serum creatinine and cystatin C levels, while a repeat sample for creatinine was taken 48 hours after. GFR was estimated using the Schwartz formula for creatinine and the Zappitelli equation for cystatin C and compared with an inulin reference. Results: The median (interquartile range [IQR]) eGFR derived from cystatin C was 48.8 (21.0) mL/min/1.73 m2, it was higher than the median creatinine-derived GFR at admission and was of statistical significance. It is also approximated closer to the inulin reference. The median (IQR) eGFR derived from creatinine at admission was 21.2 (21.4) mL/min/1.73 m2. Creatinine-derived eGFR was significantly lower in babies with asphyxia and neonatal sepsis compared to those who did not have these diagnoses. Cystatin C-derived eGFR showed no variation between the various diagnoses. Conclusion: In conclusion, cystatin C is a useful and unbiased determinant of eGFR in sick neonates as compared to creatinine.

Kidney Outcomes and Trajectories of Tubular Injury and Function in Critically Ill Patients With and Without COVID-19.

COVID-19 may injure the kidney tubules via activation of inflammatory host responses and/or direct viral infiltration. Most studies of kidney injury in COVID-19 lacked contemporaneous controls or measured kidney biomarkers at a single time point. To better understand mechanisms of acute kidney injury in COVID-19, we compared kidney outcomes and trajectories of tubular injury, viability, and function in prospectively enrolled critically ill adults with and without COVID-19. The COVID-19 Host Response and Outcomes study prospectively enrolled patients admitted to ICUs in Washington State with symptoms of lower respiratory tract infection, determining COVID-19 status by nucleic acid amplification on arrival. We evaluated major adverse kidney events (MAKE) defined as a doubling of serum creatinine, kidney replacement therapy, or death, in 330 patients after inverse probability weighting. In the 181 patients with available biosamples, we determined trajectories of urine kidney injury molecule-1 (KIM-1) and epithelial growth factor (EGF), and urine:plasma ratios of endogenous markers of tubular secretory clearance. At ICU admission, the mean age was 55 ± 16 years; 45% required mechanical ventilation; and the mean serum creatinine concentration was 1.1 mg/dL. COVID-19 was associated with a 70% greater occurrence of MAKE (relative risk 1.70; 95% CI, 1.05-2.74) and a 741% greater occurrence of KRT (relative risk 7.41; 95% CI, 1.69-32.41). The biomarker cohort had a median of three follow-up measurements. Urine EGF, secretory clearance ratios, and estimated glomerular filtration rate (eGFR) increased over time in the COVID-19 negative group but remained unchanged in the COVID-19 positive group. In contrast, urine KIM-1 concentrations did not significantly change over the course of the study in either group. Among critically ill adults, COVID-19 is associated with a more protracted course of proximal tubular dysfunction and reduced eGFR despite similar degrees of kidney injury.

Doses Evaluated in Clinical Pharmacology Studies Investigating the Effect of Intrinsic and Extrinsic Factors on PK and Safety: Case Examples from Approved Drug Development Programs.

Dose selection for investigations of intrinsic and extrinsic factors of pharmacokinetic variability as well as safety is a challenging question in the early clinical stage of drug development. The dose of an investigational product is chosen considering the compound information available to date, feasibility of the assessments, regulatory requirements, and the intent to maximize information for later regulatory submission. This review selected 37 programs as case examples of recently approved drugs to explore the doses selected with focus on studies of drug interaction, renal and hepatic impairment, food effect and concentration-QTc assessment.The review found that regulatory agencies may consider alternative approaches if justified and safe as illustrated in these examples. It is thus recommendable to use the first in human trial as an opportunity to assess QT-prolongation and drug interactions using probes or endogenous markers while maximizing the DDI potential, increasing sensitivity and ensuring safety. Early understanding of dose proportionality assists dose finding and simple and fast to conduct DDI study designs are advantageous. Single dose impairment studies despite non-proportional/time-dependent PK are often acceptability.Overall, the early understanding of the drug's safety profile is essential to ensure the safety of doses selected while preventing clinical trials with unnecessary exposure when using high doses or multiple doses. The information collected in this retrospective survey is a good reminder to tailor the early clinical program to the profile and needs of the molecule and consider regulatory opportunities to streamline the development path.

Aspartate-β-hydroxylase and hypoxia marker expression in head and neck carcinomas: implications for HPV-associated tumors

BackgroundA proportion of head and neck carcinomas (HNSCCs) are induced by high-risk human papillomaviruses (HPVs) and are associated with better patient outcomes compared to patients with HNSCCs related to tobacco and alcohol abuse. In the microenvironment of solid tumors, including HNSCCs, oxygen levels are often reduced, and a hypoxic state is induced. This can lead to a poor treatment response and a worse patient prognosis. One of the hypoxia-responsive genes is aspartate-β-hydroxylase (ASPH), whose activity promotes the growth, invasiveness, and metastasis of many types of solid tumors.MethodsIn our study, HNSCC samples were analyzed for the expression of ASPH and selected endogenous hypoxia markers by real-time PCR and/or multiplex fluorescence immunohistochemistry.ResultsExcept for the EPAS1 gene, which had higher mRNA expression in the HPV-negative group of HNSCC (p < 0.05), we found no other differences in the expression of the tested genes that were related to HPV status. On the contrary, a statistically significantly higher number of cells producing ASPH (p < 0.0001), HIF1A (p < 0.0001), GLUT1 (p < 0.0001), and MMP13 (p < 0.05) proteins were detected in the HPV-positive tumor group than in the HPV-negative sample group. All the evaluated markers, except for MMP9/13, were more abundant in the tumor parenchyma than in the tumor stroma. The Cox proportional hazard models showed that increased numbers of cells with GLUT1 and HIF1A protein expression were positive prognostic markers for overall and disease-specific survival in patients independent of HPV tumor status.ConclusionThe study examined HNSCC samples and found that elevated ASPH and hypoxia marker proteins, typically associated with poor prognosis, may actually indicate active HPV infection, the strongest prognostic factor in HNSCC patients. In cases where HPV status is uncertain, increased expression of HIF1A and GLUT1 can serve as positive prognostic factors.