Is there a role for platelet rich plasma injection in vulvar lichen sclerosus? A self-controlled pilot study.

BACKGROUND: Owing to the evidence that as many as 30-40% of patients with vulvar lichen sclerosus (VLS) fail to report a remission of symptoms with first-line corticosteroid treatment (TCS), especially as what regards dyspareunia, we aimed to analyze patients' satisfaction following vulvar injection of autologous platelet-rich plasma (PRP). This is intended as an adjunctive treatment, to be used following TCS, and appears to promote tissue repair. It may also possibly have immunomodulatory proprieties. MATERIALS AND METHODS: Patients with VLS were considered eligible for this pilot study if, despite having been treated with a 3-month TCS regimen, they reported a persistence of symptoms. PRP was produced in a referral center using a manual method and a standardized protocol. Each patient received three treatments 4 to 6 weeks apart. RESULTS: A total of 50 patients with a median age of 53 years [IQR 38-59 years] were included in the study. 6 months after the last injection of PRP all patients were either satisfied or very satisfied with the treatment (100%; 95% CI 93-100%). Median NRS scores for itching, burning, dyspareunia and dysuria were significantly reduced (p < 0.05) and FSFI, HADS and SF-12 questionnaires revealed a significant improvement in sexual function, psychological wellbeing and quality of life (p < 0.05). The number of patients reporting the need for maintenance TCS treatment was reduced by 42% (p < 0.001) and an improvement in vulvar elasticity and color was reported in all patients. CONCLUSION: Following standard medical therapy, PRP may be effective not only in improving symptoms, but also in restoring function.

Structural basis for specific inhibition of salicylate synthase from Mycobacterium abscessus.

Blocking iron uptake and metabolism has been emerging as a promising therapeutic strategy for the development of novel antimicrobial compounds. Like all mycobacteria, M. abscessus (Mab) has evolved several countermeasures to scavenge iron from host carrier proteins, including the production of siderophores, which play a crucial role in these processes. In this study, we solved, for the first time, the crystal structure of Mab-SaS, the first enzyme involved in the biosynthesis of siderophores. Moreover, we screened a small, focused library and identified a compound exhibiting a potent inhibitory effect against Mab-SaS (IC50 ≈ 2 µM). Its binding mode was investigated by means of Induced Fit Docking simulations, performed on the crystal structure presented herein. Furthermore, cytotoxicity data and pharmacokinetic predictions revealed the safety and drug-likeness of this class of compounds. Finally, the crystallographic data were used to optimize the model for future virtual screening campaigns. Taken together, the findings of our study pave the way for the identification of potent Mab-SaS inhibitors, based on both established and unexplored chemotypes.

Eco-Friendly Bio-Based Solvents for the Acetylation of the Amino Group of Amino Acids.

Nature-derived products, like juices and peel extracts of fruits and vegetables, have emerged in recent years as interesting and sustainable alternatives to traditional solvents in several synthetic applications. Herein, we present a green and fast method for the N-acetylation of amino acids, using several bio-based solvents (vinegar, tomato/kiwi/apple peel extracts, lemon juice, etc.). The high reactivity of the amino group is often a limitation in synthetic processes, making its protection a necessary step to achieve pure products and limit side reactions. Therefore, versatile, time-efficient procedures, minimal purification efforts, and good yields are desirable features for these transformations. Our new method meets all these criteria, offering a valuable and eco-friendly alternative to traditional approaches. In detail, we managed to obtain comparable yields to established setups, while improving safety and reducing the environmental impact of the overall process. Most notably, the milder conditions made it possible to avoid the use of running water (saving about 250 L/reaction) and electric-powered cooling devices.

Transfusions in autoimmune hemolytic anemias: Frequency and clinical significance of alloimmunization.

BACKGROUND: Autoimmune hemolytic anemia (AIHA) may be associated with transfusion reactions and risk of alloimmunization. OBJECTIVES: To evaluate the transfusion policy and rate of alloimmunization and its clinical significance in AIHA. METHODS: Data from 305 AIHA patients followed at a reference hematologic Center in Milan, Italy from 1997 to 2022 were retrospectively/prospectively collected (NCT05931718). RESULTS: Overall, 33% patients required transfusions with a response rate of 83% and eight transfusion reactions (7%), none hemolytic. Alloantibodies were detected in 19% of patients, being associated with higher transfusion burden (p = 0.01), lower Hb increase post-transfusion (p = 0.05), and transfusion reactions (p = 0.04). Along decades, the rate of RBC transfusions decreased from 53% to 20% and that of alloimmunization dropped from 30% to 6% likely due to the adoption of prestorage leukoreduction, the use of more restrictive Hb thresholds, and the implementation of molecular typing. CONCLUSIONS: Severe symptomatic AIHA may be safely transfused provided appropriate matching of patients and donors.

Structural Study of a New MbtI-Inhibitor Complex: Towards an Optimized Model for Structure-Based Drug Discovery.

MbtI from Mycobacterium tuberculosis (Mtb) is a Mg2+-dependent salicylate synthase, belonging to the chorismate-utilizing enzyme (CUE) family. As a fundamental player in iron acquisition, MbtI promotes the survival and pathogenicity of Mtb in the infected host. Hence, it has emerged in the last decade as an innovative, potential target for the anti-virulence therapy of tuberculosis. In this context, 5-phenylfuran-2-carboxylic acids have been identified as potent MbtI inhibitors. The first co-crystal structure of MbtI in complex with a member of this class was described in 2020, showing the enzyme adopting an open configuration. Due to the high mobility of the loop adjacent to the binding pocket, large portions of the amino acid chain were not defined in the electron density map, hindering computational efforts aimed at structure-driven ligand optimization. Herein, we report a new, high-resolution co-crystal structure of MbtI with a furan-based derivative, in which the closed configuration of the enzyme allowed tracing the entirety of the active site pocket in the presence of the bound inhibitor. Moreover, we describe a new crystal structure of MbtI in open conformation and in complex with the known inhibitor methyl-AMT, suggesting that in vitro potency is not related to the observed enzyme conformation. These findings will prove fundamental to enhance the potency of this series via rational structure-based drug-design approaches.

Unlocking the Antibiofilm Potential of Natural Compounds by Targeting the NADH:quinone Oxidoreductase WrbA.

Biofilm-dwelling cells endure adverse conditions, including oxidative imbalances. The NADH:quinone oxidoreductase enzyme WrbA has a crucial role in the mechanism of action of antibiofilm molecules such as ellagic and salicylic acids. This study aimed to exploit the potential of the WrbA scaffold as a valuable target for identifying antibiofilm compounds at non-lethal concentrations. A three-dimensional computational model, based on the published WrbA structure, was used to screen natural compounds from a virtual library of 800,000 compounds. Fisetin, morin, purpurogallin, NZ028, and NZ034, along with the reference compound ellagic acid, were selected. The antibiofilm effect of the molecules was tested at non-lethal concentrations evaluating the cell-adhesion of wild-type and WrbA-deprived Escherichia coli strains through fluorochrome-based microplate assays. It was shown that, except for NZ028, all of the selected molecules exhibited notable antibiofilm effects. Purpurogallin and NZ034 showed excellent antibiofilm performances at the lowest concentration of 0.5 µM, in line with ellagic acid. The observed loss of activity and the level of reactive oxygen species in the mutant strain, along with the correlation with terms contributing to the ligand-binding free energy on WrbA, strongly indicates the WrbA-dependency of purpurogallin and NZ034. Overall, the molecular target WrbA was successfully employed to identify active compounds at non-lethal concentrations, thus revealing, for the first time, the antibiofilm efficacy of purpurogallin and NZ034.

Mechanistic Insights into the Antibiofilm Mode of Action of Ellagic Acid.

Bacterial biofilm is a major contributor to the persistence of infection and the limited efficacy of antibiotics. Antibiofilm molecules that interfere with the biofilm lifestyle offer a valuable tool in fighting bacterial pathogens. Ellagic acid (EA) is a natural polyphenol that has shown attractive antibiofilm properties. However, its precise antibiofilm mode of action remains unknown. Experimental evidence links the NADH:quinone oxidoreductase enzyme WrbA to biofilm formation, stress response, and pathogen virulence. Moreover, WrbA has demonstrated interactions with antibiofilm molecules, suggesting its role in redox and biofilm modulation. This work aims to provide mechanistic insights into the antibiofilm mode of action of EA utilizing computational studies, biophysical measurements, enzyme inhibition studies on WrbA, and biofilm and reactive oxygen species assays exploiting a WrbA-deprived mutant strain of Escherichia coli. Our research efforts led us to propose that the antibiofilm mode of action of EA stems from its ability to perturb the bacterial redox homeostasis driven by WrbA. These findings shed new light on the antibiofilm properties of EA and could lead to the development of more effective treatments for biofilm-related infections.

Biological Screening and Crystallographic Studies of Hydroxy γ-Lactone Derivatives to Investigate PPARγ Phosphorylation Inhibition.

PPARγ represents a key target for the treatment of type 2 diabetes and metabolic syndrome. To avoid serious adverse effects related to the PPARγ agonism profile of traditional antidiabetic drugs, a new opportunity is represented by the development of molecules acting as inhibitors of PPARγ phosphorylation by the cyclin-dependent kinase 5 (CDK5). Their mechanism of action is mediated by the stabilization of the PPARγ ß-sheet containing Ser273 (Ser245 in PPARγ isoform 1 nomenclature). In this paper, we report the identification of new γ-hydroxy-lactone-based PPARγ binders from the screening of an in-house library. These compounds exhibit a non-agonist profile towards PPARγ, and one of them prevents Ser245 PPARγ phosphorylation by acting mainly on PPARγ stabilization and exerting a weak CDK5 inhibitory effect.

Anemia management and transfusion strategy in internal medicine units: Less is more.

Blood transfusion is one of the most overused procedures, especially in elderly patients. Despite the current transfusion guidelines recommending a restrictive transfusion strategy in stable patients, the clinical practice varies according to physicians' experience and implementation of patient blood management. This study aimed to evaluate the anemia management and transfusion strategy in anemic elderly hospitalized and the impact of an educational program. We enrolled ≥ 65-year-old patients who presented or developed anemia during admission to a tertiary hospital's internal medicine and geriatric units. Patients with onco-hematological disorders, hemoglobinopathies and active bleeding were excluded. In the first phase, anemia management was monitored. In the second phase, the six participating units were divided into two groups and two arms: Educational (Edu) and non-educational (NE). During this phase, physicians in the Edu arm underwent an educational program for the appropriate use of transfusion and anemia management. In the third phase, anemia management was monitored. Comorbidities, demographic and hematological characteristics were similar in all phases and arms. The percentages of transfused patients during phase 1 were 27.7% in NE and 18.5% in the Edu arm. During phase 3, it decreased to 21.4% in the NE and 13.6% in the Edu arm. Hemoglobin levels at discharge and after 30 days were higher in the Edu group despite reduced use of blood transfusion. In conclusion, a more restrictive strategy was comparable or superior to the more liberal one in terms of clinical outcomes, with the advantage of saving red blood cell units and reducing related side effects.

Iron Acquisition and Metabolism as a Promising Target for Antimicrobials (Bottlenecks and Opportunities): Where Do We Stand?

The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) infections is one of the most crucial challenges currently faced by the scientific community. Developments in the fundamental understanding of their underlying mechanisms may open new perspectives in drug discovery. In this review, we conducted a systematic literature search in PubMed, Web of Science, and Scopus, to collect information on innovative strategies to hinder iron acquisition in bacteria. In detail, we discussed the most interesting targets from iron uptake and metabolism pathways, and examined the main chemical entities that exhibit anti-infective activities by interfering with their function. The mechanism of action of each drug candidate was also reviewed, together with its pharmacodynamic, pharmacokinetic, and toxicological properties. The comprehensive knowledge of such an impactful area of research will hopefully reflect in the discovery of newer antibiotics able to effectively tackle the antimicrobial resistance issue.

Targeting Siderophore-Mediated Iron Uptake in M. abscessus: A New Strategy to Limit the Virulence of Non-Tuberculous Mycobacteria.

Targeting pathogenic mechanisms, rather than essential processes, represents a very attractive approach for the development of new antimycobacterial drugs. In this context, iron acquisition routes have recently emerged as potentially druggable pathways. However, the importance of siderophore biosynthesis in the virulence and pathogenicity of M. abscessus (Mab) is still poorly understood. In this study, we investigated the Salicylate Synthase (SaS) of Mab as an innovative molecular target for the development of inhibitors of siderophore production. Notably, Mab-SaS does not have any counterpart in human cells, making it an interesting candidate for drug discovery. Starting from the analysis of the binding of a series of furan-based derivatives, previously identified by our group as inhibitors of MbtI from M. tuberculosis (Mtb), we successfully selected the lead compound 1, exhibiting a strong activity against Mab-SaS (IC50 ≈ 5 µM). Computational studies characterized the key interactions between 1 and the enzyme, highlighting the important roles of Y387, G421, and K207, the latter being one of the residues involved in the first step of the catalytic reaction. These results support the hypothesis that 5-phenylfuran-2-carboxylic acids are also a promising class of Mab-SaS inhibitors, paving the way for the optimization and rational design of more potent derivatives.

BORN study: a multicenter randomized trial investigating cord blood red blood cell transfusions to reduce the severity of retinopathy of prematurity in extremely low gestational age neonates.

BACKGROUND: Extremely low gestational age neonates (ELGANs, i.e., neonates born before 28 weeks of gestation) are at high risk of developing retinopathy of prematurity (ROP), with potential long-life visual impairment. Due to concomitant anemia, ELGANs need repeated red blood cell (RBC) transfusions. These produce a progressive replacement of fetal hemoglobin (HbF) by adult hemoglobin (HbA). Furthermore, a close association exists between low levels of HbF and severe ROP, suggesting that a perturbation of the HbF-mediated oxygen release may derange retinal angiogenesis and promote ROP. METHODS/DESIGN: BORN (umBilical blOod to tRansfuse preterm Neonates) is a multicenter double-blinded randomized controlled trial in ELGANs, to assess the effect of allogeneic cord blood RBC transfusions (CB-RBCs) on severe ROP development. Recruitment, consent, and randomization take place at 10 neonatology intensive care units (NICUs) of 8 Italian tertiary hospitals. ELGANs with gestational age at birth comprised between 24+0 and 27+6 weeks are randomly allocated into two groups: (1) standard RBC transfusions (adult-RBCs) (control arm) and (2) CB-RBCs (intervention arm). In case of transfusion need, enrolled patients receive transfusions according to the allocation arm, unless an ABO/RhD CB-RBC is unavailable. Nine Italian public CB banks cooperate to make available a suitable amount of CB-RBC units for all participating NICUs. The primary outcome is the incidence of severe ROP (stage 3 or higher) at discharge or 40 weeks of postmenstrual age, which occurs first. DISCUSSION: BORN is a groundbreaking trial, pioneering a new transfusion approach dedicated to ELGANs at high risk for severe ROP. In previous non-randomized trials, this transfusion approach was proven feasible and able to prevent the HbF decrease in patients requiring multiple transfusions. Should the BORN trial confirm the efficacy of CB-RBCs in reducing ROP severity, this transfusion strategy would become the preferential blood product to be used in severely preterm neonates. TRIAL REGISTRATION: ClinicalTrials.gov NCT05100212. Registered on October 29, 2021.

Dampening of cytotoxic innate lymphoid cells: A new tumour immune escape mechanism in B cell non-Hodgkin's lymphoma.

The role and regulation of innate immune cells is poorly understood in B-cell non-Hodgkin lymphoma (NHL). As natural killer (NK) cells, helper innate lymphoid cells (ILCs) are lymphocytes endowed with either anti- or pro-tumour activity and involved in inflammatory processes. In our ex vivo analysis of NK cells and ILCs from NHL patients, we observed that, in comparison to healthy donors (HD), the frequency of the cytotoxic subset of NK cells, the CD16+ NK, decreased in patients' peripheral blood. In general, circulating NK cells showed a pro-tumorigenic phenotype, while ILCs displayed a more activated/cytotoxic phenotype. Conversely, at the tumour site, in patients' lymph nodes, ILCs showed a low expression of granzyme.In vitromixed lymphocyte-tumour cell cultures with HD PBMCs and NHL cell lines demonstrated that ILC cytotoxic potential was lowered by the presence of tumour cells but, in the absence of T regulatory cells (Tregs), their cytolytic potential was recovered. Our data shed novel light on dysfunctional innate immunity in NHL. We suggest a new mechanism of tumour immuno-escape based on the reduction of cell cytotoxicity involving ILCs and likely controlled by Tregs.

Veno-Arterial Extracorporeal Membrane Oxygenation (ECMO) Impairs Bradykinin-Induced Relaxation in Neonatal Porcine Coronary Arteries.

Extracorporeal membrane oxygenation (ECMO) is a lifesaving support for respiratory and cardiovascular failure. However, ECMO induces a systemic inflammatory response syndrome that can lead to various complications, including endothelial dysfunction in the cerebral circulation. We aimed to investigate whether ECMO-associated endothelial dysfunction also affected coronary circulation. Ten-day-old piglets were randomized to undergo either 8 h of veno-arterial ECMO (n = 5) or no treatment (Control, n = 5). Hearts were harvested and coronary arteries were dissected and mounted as 3 mm rings in organ baths for isometric force measurement. Following precontraction with the thromboxane prostanoid (TP) receptor agonist U46619, concentration−response curves to the endothelium-dependent vasodilator bradykinin (BK) and the nitric oxide (NO) donor (endothelium-independent vasodilator) sodium nitroprusside (SNP) were performed. Relaxation to BK was studied in the absence or presence of the NO synthase inhibitor Nω-nitro-L-arginine methyl ester HCl (L-NAME). U46619-induced contraction and SNP-induced relaxation were similar in control and ECMO coronary arteries. However, BK-induced relaxation was significantly impaired in the ECMO group (30.4 ± 2.2% vs. 59.2 ± 2.1%; p < 0.0001). When L-NAME was present, no differences in BK-mediated relaxation were observed between the control and ECMO groups. Taken together, our data suggest that ECMO exposure impairs endothelium-derived NO-mediated coronary relaxation. However, there is a NO-independent component in BK-induced relaxation that remains unaffected by ECMO. In addition, the smooth muscle cell response to exogenous NO is not altered by ECMO exposure.

Synthesis and Assessment of the In Vitro and Ex Vivo Activity of Salicylate Synthase (Mbti) Inhibitors as New Candidates for the Treatment of Mycobacterial Infections.

Tuberculosis (TB) causes millions of deaths every year, ranking as one of the most dangerous infectious diseases worldwide. Because several pathogenic strains of M. tuberculosis (Mtb) have developed resistance against most of the established anti-TB drugs, new therapeutic options are urgently needed. An attractive target for the development of new anti-TB agents is the salicylate synthase MbtI, the first enzyme of the mycobacterial siderophore biochemical machinery, absent in human cells. In this work, a set of analogues of 5-(3-cyanophenyl)furan-2-carboxylic acid (I), the most potent MbtI inhibitor identified to date, was synthesized, characterized, and tested to further elucidate the structural requirements for achieving an efficient MbtI inhibition and potent antitubercular activity. The structure-activity relationships (SAR) discussed herein evidenced the importance of the side chain linked to the phenyl moiety to improve the in vitro antimycobacterial activity. In detail, 1f emerged as the most effective analogue against the pathogen, acting without cytotoxicity issues. To deepen the understanding of its mechanism of action, we established a fluorescence-based screening test to quantify the pathogen infectivity within host cells, using MPI-2 murine cells, a robust surrogate for alveolar macrophages. The set-up of the new assay demonstrates significant potential to accelerate the discovery of new anti-TB drugs.

Insights on the Modulation of SIRT5 Activity: A Challenging Balance.

SIRT5 is a member of the Sirtuin family, a class of deacetylating enzymes consisting of seven isoforms, involved in the regulation of several processes, including gene expression, metabolism, stress response, and aging. Considering that the anomalous activity of SIRT5 is linked to many pathological conditions, we present herein an overview of the most interesting modulators, with the aim of contributing to further development in this field.

Virtual screening and crystallographic studies reveal an unexpected γ-lactone derivative active against MptpB as a potential antitubercular agent.

Mycobacterial resistance is a rapidly increasing phenomenon requiring the identification of new drugs effective against multidrug-resistant pathogens. The inhibition of protein tyrosine phosphatase B (MptpB), which interferes with host immune responses, may provide a new strategy to fight tuberculosis (TB), while preventing cross-resistance issues. On this basis, starting from a virtual screening (VS) campaign and subsequent structure elucidation studies guided by X-ray analyses, an unexpected γ-lactone derivative (compound 1) with a significant enzymatic activity against MptpB was identified. The structural characterization of compound 1 was described by means of NMR spectroscopy, HRMS, single crystal X-ray diffraction and Hirshfeld surface analysis, allowing a detailed conformational investigation. Notably, the HPLC separation of (±)-1 led to the isolation of the most active isomer, which emerged as a very promising MptpB inhibitor, with an IC50 value of 31.1 µM. Overall, the new chemotype described herein might serve as a basis for the development of novel treatments against TB infections.

An Outline of the Latest Crystallographic Studies on Inhibitor-Enzyme Complexes for the Design and Development of New Therapeutics against Tuberculosis.

The elucidation of the structure of enzymes and their complexes with ligands continues to provide invaluable insights for the development of drugs against many diseases, including bacterial infections. After nearly three decades since the World Health Organization's (WHO) declaration of tuberculosis (TB) as a global health emergency, Mycobacterium tuberculosis (Mtb) continues to claim millions of lives, remaining among the leading causes of death worldwide. In the last years, several efforts have been devoted to shortening and improving treatment outcomes, and to overcoming the increasing resistance phenomenon. The structural elucidation of enzyme-ligand complexes is fundamental to identify hot-spots, define possible interaction sites, and elaborate strategies to develop optimized molecules with high affinity. This review offers a critical and comprehensive overview of the most recent structural information on traditional and emerging mycobacterial enzymatic targets. A selection of more than twenty enzymes is here discussed, with a special emphasis on the analysis of their binding sites, the definition of the structure-activity relationships (SARs) of their inhibitors, and the study of their main intermolecular interactions. This work corroborates the potential of structural studies, substantiating their relevance in future anti-mycobacterial drug discovery and development efforts.

Cord Blood Platelet Gel as a Treatment of Occipital Pressure Injuries in Newborns: Report of Two Cases.

BACKGROUND: A Pressure Ulcer (PU) is a severe event and could create discomfort to newborns. In newborns, one of mostly stricken location by PU is occipital area. Recent studies have highlighted that Cord Blood Platelet Gel (CBPG) might be a better alternative compared to traditional treatment. We report two cases of occipital PU treated with CBPG. CASE REPORT: Two male infants showing occipital PU were treated with standard local treatment, but no improvement was observed. After parental informed consent was obtained, CBPG application on PU was performed every 48 h. In these two cases of PU, a fast improvement in healing was observed since the first application of CBPG. The PU healed resulted in a scar after 53 and 50 days (Case 1 and Case 2, respectively) from development. No complications or infections were reported. CONCLUSIONS: CBPG contains many angiogenetic and growth factors, these characteristics make it indicated in treating soft tissue injuries. It would seem to be safe and an effective treatment of neonatal PUs reducing the time of the healing and the hospitalization and the infectious risks. Further studies are needed to evaluate long term aesthetic and functional results of PU treated with CBPG.

Natural products against key Mycobacterium tuberculosis enzymatic targets: Emerging opportunities for drug discovery.

For centuries, natural products (NPs) have served as powerful therapeutics against a variety of human ailments. Nowadays, they still represent invaluable resources for the treatment of many diseases, including bacterial infections. After nearly three decades since the World Health Organization's (WHO) declaration of tuberculosis (TB) as a global health emergency, Mycobacterium tuberculosis (Mtb) continues to claim millions of lives, remaining among the leading causes of death worldwide. In the last years, several efforts have been devoted to shortening and improving treatment outcomes, and to overcoming the increasing resistance phenomenon. Nature has always provided a virtually unlimited source of bioactive molecules, which have inspired the development of new drugs. NPs are characterized by an exceptional chemical and structural diversity, the result of millennia of evolutionary responses to various stimuli. Thanks to their favorable structural features and their enzymatic origin, they are naturally prone to bind proteins and exhibit bioactivities. Furthermore, their worldwide distribution and ease of accessibility has contributed to promote investigations on their activity. Overall, these characteristics make NPs excellent models for the design of novel therapeutics. This review offers a critical and comprehensive overview of the most promising NPs, isolated from plants, fungi, marine species, and bacteria, endowed with inhibitory properties against traditional and emerging mycobacterial enzymatic targets. A selection of 86 compounds is here discussed, with a special emphasis on their biological activity, structure-activity relationships, and mechanism of action. Our study corroborates the antimycobacterial potential of NPs, substantiating their relevance in future drug discovery and development efforts.