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Bacterial infections and multidrug-resistant bacteria are major health burdens in wound care. Biocompatible antimicrobial agents, e.g., ε-polylysine (ε-PL), provide a broad spectrum of antibacterial properties and support dermal cell growth. Here, ε-PL was incorporated into polycaprolactone (PCL)/gelatin electrospun scaffolds collected at varying rotation speeds. Then, the samples were crosslinked using dopamine hydrochloride to provide highly proliferative dressings with broad antimicrobial activity. The morphological study showed that the electrospun wound dressings were smooth, continuous, and bead-free, with a mean diameter ranging from 267 ± 7 to 331 ± 8 nm for all random and aligned nanofibers. The fiber alignment of the electrospun PCL/gelatin scaffolds improved their tensile strength and modulus. Moreover, nanofiber mats are highly hydrophilic, which is crucial for an efficient wound dressing. The samples also demonstrated high antimicrobial properties against common wound bacterial strains, including methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus aureus (SA), Escherichia coli (EC), Acinetobacter baumannii (AB), and Pseudomonas aeruginosa (PA). Mammalian cell proliferation and morphology assays involving primary human dermal fibroblasts (hDFs) and immortalized keratinocytes (HaCaT) showed excellent biocompatibility of the electrospun mats and remarkably aligned mats. Furthermore, aligned mats showed more cell migration than randomly oriented mats, which is desirable for more efficient wound healing. Therefore, it can be concluded that aligned PCL/gelatin mats containing ε-PL are promising for potential use in wound dressings. Graphical Abstract: [Figure not available: see fulltext.]. Agency for Science, Technology and Research (A*STAR) Ministry of Education (MOE) Nanyang Technological University E.R.G. acknowledges the financial support from the Singapore International Graduate Award (SINGA). R.L. thanks funding support from the Duke-NUS Khoo Bridge Funding Award (Duke-NUS-KBrFA/2021/0044). N.K.V. acknowledges funding support from the Singapore Ministry of Education (MOE) under its MOE Academic Research Fund (AcRF) Tier 1 Grant (RG26/20) and the Agency for Science, Technology and Research (A*STAR) under its Wound Care Innovation for the Tropics (WCIT) Industry Alignment Fund Pre-Positioning (IAF-PP) Grant (H17/01/a0/0K9). B.H.S.W. and Z.S.P. were provided Ph.D. fellowships by HealthTech NTU and Lee Kong Chian School of Medicine, Nanyang Technological University Singapore.

Bacteriophages and phage-derived proteins are a promising class of antibacterial agents that experience a growing worldwide interest. To map ongoing phage research in Singapore and neighboring countries, Lee Kong Chian School of Medicine, Nanyang Technological University Singapore (NTU) and Yong Loo Lin School of Medicine, National University of Singapore (NUS) recently co-organized a virtual symposium on Bacteriophage and Bacteriophage-Derived Technologies, which was attended by more than 80 participants. Topics were discussed relating to phage life cycles, diversity, the roles of phages in biofilms and the human gut microbiome, engineered phage lysins to combat polymicrobial infections in wounds, and the challenges and prospects of clinical phage therapy. This perspective summarizes major points discussed during the symposium and new perceptions that emerged after the panel discussion. Agency for Science, Technology and Research (A*STAR) Ministry of Health (MOH) National Medical Research Council (NMRC) National Research Foundation (NRF) Following supports are acknowledged: N.K.V. to LeeKong Chian School of Medicine Strategic Academic In-itiative Grant (L0494003); H.C. to A*STAR Grant(C210812044); P.H., B.C.G., and W.M. to Singapore Na-tional Research Foundation (NRF)’s Intra-Campus for Re-search Excellence and Technological Enterprise (CREATE)Seed Collaboration Grant; P.H. and B.C.G. to Singapore-MIT Alliance for Research and Technology (SMART) An-timicrobial Resistance Interdisciplinary Group funded by theNRF under its CREATE program; T.A.A. to NUSMedpostdoctoral fellowship (NUHSRO/2019/046/PDF/19); J.P.B.to National University Hospital System Seed Fund (NUHSRO/2019/050/T1/Seed-Mar/03) and SMART (ING-001014 BIO);W.M. to Singapore Ministry of Health’s National MedicalResearch Council (OFIRG21jun-0038); M.A.M.V.S. to Na-tional Health Innovation Centre Singapore Innovation to De-velop Grant (NHIC-I2D-2104625).

Burn wounds are susceptible to microbial invasion from both resident and exogenous bacteria, which becomes a critical public health issue and causes substantial economic burden. There is a perceived demand to produce new antimicrobial wound dressings that hinder bacterial colonization while accelerating the healing process and hence would provide an improved standard of care for patients. Since ancient times, herbal extracts from medicinally important plants have extensively been used for treating burn injuries. This work reports the utility of electrospun nanofibers containing plant extracts and antibiotics combination as a multifunctional scaffold for treating second-degree burns. First, we determined the various components of plant extracts from Gymnema sylvestre by two different processing methods and their synergism with minocycline antibiotics. Then, we prepared core-shell nanofibrous dressings with poly-e-caprolactone/gelatin laden with minocycline hydrochloride as a shell and gelatin infused with G. sylvestre extracts (ultrasound-assisted extracts and cold macerated extracts) as the core using coaxial electrospinning. The electrospun nanofibers displayed a smooth, continuous, and bead-free morphology with adequate wettability. The presence of extract components in the core-shell nanofibers resulted in enhanced mechanical properties when compared to pristine mats. The core-shell structures resulted in sustained release of the bioactive components when compared to nanofiber blends. Core-shell nanofiber mats containing plant extracts and antibiotic combinations displayed potent antimicrobial and antibiofilm properties while promoting the spread and proliferation of skin cells when compared to pristine mats. In a porcine model of cutaneous second-degree burns, we showed that wounds treated with the antimicrobial dressing improved re-epithelialization and collagen organization in comparison to untreated wounds.

Background: Hesperetin is a natural compound known for its cholesterol-lowering effect and a wide range of pharmacological activities. Objectives: Investigating the potential anticancer activities of Hesperetin in malignant hematolymphoid cell lines HuT78 and MJ, derived from patients with Cutaneous T-Cell Lymphomas (CTCL). Methods: The cytotoxic effect of Hesperetin on two different CTCL cell lines, HuT78 and MJ, was assessed by MTS-based colorimetric assay. Apoptosis, cell cycle, ROS (Reactive Oxygen Species) and molecular analysis were performed using flow-cytometry and immunoblotting. Results: Hesperetin-treated CTCL cells were arrested at the sub-G1 phase of cell cycle with the concomitant decrease in the expression of the cell cycle regulator protein cyclin B. In addition, the study found that the cellular treatment with Hesperetin caused an induction of apoptosis, which was independent of ROS generation. Hesperetin caused a significant decrease in the expression level of anti-apoptotic protein Bcl-xL and an increase in cleaved caspase-3 and PARP proteins in CTCL cells. Furthermore, Hesperetin treatment in CTCL cells down-regulated the expression of Notch1 and phosphorylation of STAT3 (Tyr705) and inhibited NFκBp65. Conclusion: This study highlights the anticancer properties of Hesperetin. Which induces apoptosis in CTCL cells via STAT3/Notch1/NFκB mediated signaling pathway, suggesting that further development of this novel class of flavonoid may contribute to new drug discovery for certain hematolymphoid malignancies.

Here, we present a rational approach that enhances the membrane selectivity of a prolific pore-forming peptide, melittin, based on experimental observations that the cationic polymer, e-polylysine, disrupts bacterial membranes with greater affinity over mammalian cells when compared to poly-l-lysine and poly-d-lysine. We systematically replaced three α-lysine residues in melittin with e-lysine residues and identified key residues that are important for cytotoxicity. We then assessed the antimicrobial properties of the modified peptides which carry two or three e-lysyl residues. Two modified melittin peptides displayed rapid bactericidal properties against antibiotic-resistant strains, low innate resistance development by pathogenic bacteria, remained nonimmunogenic for T lymphocytes, and increased bioavailability in tear fluids. In proof-of-concept in vivo experiments, one of the peptides was noncytotoxic for ocular surfaces and had comparable antimicrobial efficacy to that of fluoroquinolone antibiotics. The results uncover a simple and potential strategy that can enhance the membrane selectivity of cytolytic peptides by e-lysylation.

The development of antimicrobial nanofibre dressings that can protect the injured tissues from commensal pathogens while promoting tissue regeneration finds enormous potential in plastic and reconstructive surgery practices. To achieve this goal, we investigated the effect of chondroitin sulphate on the morphology, mechanical properties, wettability and biocompatibility of polydopamine crosslinked electrospun gelatin nanofibres containing mineralized magnesium. To extend the durability of dressings, we prepared composite dressings containing polycaprolactone (PCL) and gelatin as blend or core-shell nanofibres. Nanofibre blends presented greater tensile strength and stretchability, while core-shell nanofibres displayed superior photoluminescent properties. In a porcine model of cutaneous burn injury, both the blend and core-shell nanofibre dressings displayed improved re-epithelialization, wound closure and clinical outcome in comparison to untreated burns. Histology of the biopsied tissues indicated smooth regeneration and collagen organization of the burns treated with core-shell nanostructures than untreated burns. This study compared the physico-chemical and biological properties of composite nanofibres that are capable of accelerating burn wound healing and possess antimicrobial properties, highlighting their potential as wound dressings and skin substitutes.

Antibacterial properties of copper have been known for ages. With the rise of antimicrobial resistance (AMR), hospital-acquired infections, and the current SARS-CoV-2 pandemic, copper and copper-derived materials are being widely researched for healthcare ranging from therapeutics to advanced wound dressing to medical devices. We cover current research that highlights the potential uses of metallic and ionic copper, copper alloys, copper nanostructures, and copper composites as antibacterial, antifungal, and antiviral agents, including those against the SARS-CoV-2 virus. The applications of copper-enabled engineered materials in medical devices, wound dressings, personal protective equipment, and self-cleaning surfaces are discussed. We emphasize the potential of copper and copper-derived materials in combating AMR and efficiently reducing infections in clinical settings.

Non-Hodgkin a diverse group of malignancies, encompassing the common difuse large B-cell lymphoma (DLBCL) to the rarer T-cell lymphomas. Chemoresistance is a major barrier to treatment and the mechanisms through which it occurs are incompletely understood [1]. Although eforts are made to target frequently dysregulated pathways in NHL subtypes, these diseases still evolve into aggressive forms resistant even to newer therapies [2].

Background: Dying tumor cells release intracellular potassium (K(+)), raising extracellular K(+) ([K(+)](e)) in the tumor microenvironment (TME) to 40–50 mM (high-[K(+)](e)). Here, we investigated the effect of high-[K(+)](e) on T cell functions. Materials and Methods: Functional impacts of high-[K(+)](e) on human T cells were determined by cellular, molecular, and imaging assays. Results: Exposure to high-[K(+)](e) suppressed the proliferation of central memory and effector memory T cells, while T memory stem cells were unaffected. High-[K(+)](e) inhibited T cell cytokine production and dampened antitumor cytotoxicity, by modulating the Akt signaling pathway. High-[K(+)](e) caused significant upregulation of the immune checkpoint protein PD-1 in activated T cells. Although the number of K(Ca)3.1 calcium-activated potassium channels expressed in T cells remained unaffected under high-[K(+)](e), a novel K(Ca)3.1 activator, SKA-346, rescued T cells from high-[K(+)](e)-mediated suppression. Conclusion: High-[K(+)](e) represents a so far overlooked secondary checkpoint in cancer. K(Ca)3.1 activators could overcome such “ionic-checkpoint”-mediated immunosuppression in the TME, and be administered together with known PD-1 inhibitors and other cancer therapeutics to improve outcomes.

Developing an effective antibacterial surface with the help of drugs that prevent bacterial adhesion, colonization, and proliferation into the surrounding tissues is of great demand. Rifampicin (Rf) is effective antibiotic drug proved has proved its potential in treating bacteria in biofilms, especially against the microbes causing bone infections. Hydroxyapatite (HA), a biocompatible osteoconductive ceramic, has been verified to be a significant material for bioactivity enhancement. Electrospinning is an effective inexpensive method for incorporating nanoparticles into nanofibers with uniform distribution for the drug delivery system for tissue engineering applications. In the current study, for improving bioactivity and antibacterial properties, novel functional polycaprolactone (PCL) composite nanofibers loaded HA and Rf was developed and coated on titanium (Ti). Different characterization techniques such as SEM, EDS, XRD, FITR were used to analyze these PCL/Rf/HA nanocomposites. The results showed that the bioactivity and tensile strength of the composite scaffold increased with the addition of HA nanoparticles. In vitro bioactivity demonstrated that the PCL/HA/Rf composite nanofibers possess enhanced calcium deposition when compared to the pure sample. Cellular interactive responses such as adhesive and proliferation were evaluated using hFOB human fetal osteoblast cell lines. After 6 days of culturing, the cellular properties on Ti sample coated with PCL/HA/Rf was significantly improved. Antibacterial evaluations on the substrates showed that Rf-loaded PCL/HA fibers displayed >3 log reduction against S.aureus MRSA, and S.epidermidis bacterial strain and >2 log reduction against P.aeruginosa bacteria. In vitro drug release study shows initial burst release of Rf, followed by sustained released of 62% at the end of 32 days. The cell viability, adhesion, and proliferation evaluation suggest that the PCL/HA/Rf coated substrate possess good cytocompatibility. Further incorporation of Rf enhanced the antibacterial property of this nanofibrous scaffold.

Background:Dying tumor cells release intracellular potassium (K+), raising extracellular K+([K+]e) in the tumor microenvironment (TME) to 40–50 mM (high-[K+]e). Here, we investigated the effect of high-[K+]eon T cell functions. Materials and Methods:Functional impacts of high-[K+]eon human T cells were determined by cellular, molecular, and imaging assays. Results:Exposure to high-[K+]esuppressed the proliferation of central memory and effector memory T cells, while T memory stem cells were unaffected. High-[K+]einhibited T cell cytokine production and dampened antitumor cytotoxicity, by modulating the Akt signaling pathway. High-[K+]ecaused significant upregulation of the immune checkpoint protein PD-1 in activated T cells. Although the number of KCa3.1 calcium-activated potassium channels expressed in T cells remained unaffected under high-[K+]e, a novel KCa3.1 activator, SKA-346, rescued T cells from high-[K+]e-mediated suppression. Conclusion:High-[K+]erepresents a so far overlooked secondary checkpoint in cancer. KCa3.1 activators could overcome such “ionic-checkpoint”-mediated immunosuppression in the TME, and be administered together with known PD-1 inhibitors and other cancer therapeutics to improve outcomes.

The nanostructured titanium (Ti) obtained by the equal-channel angular pressing (ECAP) has shown great promise as a biomedical implant material over the past few decades. The present work aims to investigate the effect of topographical changes caused by ECAP and piranha treatment (Tr) on the surface performance and biological properties of Ti for bone tissue engineering applications. The effects of dual treatments, i.e., ECAP and Tr, on Ti were systematically investigated by multiple characterization techniques, surface wettability, apatite-forming ability, cellular behavior, and antibacterial studies. We demonstrate that both ECAP and ECApTr samples possess desirable mechanical and physical properties and are biocompatible to cultured human fetal osteoblast (hFOB) cells. The potential of adhesion and proliferation of hFOB cells on ECAP and ECApTr samples was found to be superior to that of control unprocessed sample (annealed). Ti samples prepared by both methods showed excellent antimicrobial properties against clinical strains of the most common pathogenic bacteria causing orthopedic implant infections, Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa). This study supports the established claim about mechanical properties improvement by ultrafine refinement and further enhances the antibacterial properties when chemically etched with a piranha solution.

Carbonic anhydrases (CAs) are ubiquitously expressed enzymes and catalyse an important physiological reaction of interconverting the hydration of carbon dioxide to bicarbonates, which is crucial for maintaining acid/base equilibrium in certain tissues. While 15 different isoforms of CAs are present in various cell types in human tissues, their expression pattern in the epidermis remains to be investigated. Here, we report the expression of 5 CA isoforms (CAII, CAIX, CAXI, CAXII and CAXIII) in human primary keratinocytes. Further, we demonstrate that the expression of CAII and CAIX in these cells is significantly up-regulated by the biologically active metabolites of vitamin A (all-trans retinoic acid) and vitamin D (1α,25-dihydroxyvitamin D3), respectively. Taken together, apart from providing new information on the expression of CAs in the skin, our results highlight a previously undisclosed connection between vitamin A and CAII expression and vitamin D and CAIX expression. (Less)

Increased evolution of multidrug resistant pathogens necessitates the development of multifunctional antimicrobials. There is a perceived need for developing new antimicrobials that can interfere with acute inflammation after bacterial infections. Here, we investigated the therapeutic potential of linear polyethylenimine (LPEI) in vitro and in vivo. The minimum inhibitory concentration of LPEI ranged from 8 to 32 μg/mL and elicited rapid bactericidal activity against clinical isolates of meticillin-resistant Staphylococcus aureus (MRSA). The polymer was biocompatible for human cultured ocular and dermal cells. Prophylactic addition of LPEI inhibited the bacterial colonization of human primary dermal fibroblasts (hDFs). In a scratch wound cell migration assay, LPEI attenuated the migration inhibitory effects of bacterial secretions. The polymer neutralized the cytokine release by hDFs exposed to bacterial secretions, possibly by blocking their accessibility to host cell receptors. Topical instillation of LPEI (1 mg/mL) was noncytotoxic and did not affect the re-epithelialization of injured porcine cornea. In a prophylactic in vivo model of S. aureus keratitis, LPEI was superior to gatifloxacin in terms of reducing stimulation of cytokines, corneal edema, and overall severity of the infection. These observations demonstrate therapeutic potential of LPEI for antimicrobial prophylaxis.

Non-Hodgkin’s lymphoma (NHL) is a diverse group of malignancies, encompassing the most common diffuse large B-cell lymphoma (DLBCL) to the rarer T-cell lymphomas. DDX3X is an RNA helicase and, depending on tumour type, plays tumour suppressive or oncogenic roles in cancer. However, its role in NHL is not clear. Targeted sequencing of DDX3X hotspots on exons 8-15 in a cohort of 158 unselected DLBCL subjects showed DDX3X mutations in 5 cases; whereas whole exome sequencing in a cohort of 9 relapsed/refractory DLBCL patients treated with R-CHOP identified DDX3X mutations in 4 cases. DLBCL patients (n=223) with DDX3X mutations had worse 5-year overall survival (22%) compared to patients with wild-type DDX3X (72%, p=0.021). Using DLBCL and cutaneous T-cell lymphoma (CTCL) cell lines, we showed that the expression of mutant DDX3X-R475C or DDX3X knockdown significantly enhanced cell migratory/invasive potential and elevated the phosphorylation of STAT3, Akt and p42/44. DDX3X loss increased resistance to doxorubicin and histone deacetylase targeting drugs in DLBCL and CTCL cells, respectively. Importantly, B- and T-cell lineage DDX3X-depleted cells remained sensitive to STAT3 inhibition. We conclude that DDX3X mutations are important driver lesions in NHL subtypes and are associated with chemoresistance but may be countered with a STAT3 inhibitor.

Simple Summary Glucocorticoid therapy resistance in B-cell malignancies is often associated with constitutive activation of tyrosine kinases. Novel anticancer drugs targeting hyperactivated tyrosine kinases, such as Bruton's tyrosine kinase (BTK), have, therefore, gained much interest over the past few decades and have already been approved for clinical use. In this study, we compared the therapeutic efficacy of the phytochemical kinase inhibitor withaferin A with the clinically approved BTK inhibitor ibrutinib to target hyperactivated tyrosine kinase signaling in glucocorticoid-resistant multiple myeloma cells. Our results demonstrate that withaferin A-induced cell death of glucocorticoid-resistant MM1R cells involves covalent cysteine targeting of multiple Hinge-6 domain type tyrosine kinases of the kinase cysteinome classification, including BTK. Multiple myeloma (MM) is a hematological malignancy characterized by plasma cells' uncontrolled growth. The major barrier in treating MM is the occurrence of primary and acquired therapy resistance to anticancer drugs. Often, this therapy resistance is associated with constitutive hyperactivation of tyrosine kinase signaling. Novel covalent kinase inhibitors, such as the clinically approved BTK inhibitor ibrutinib (IBR) and the preclinical phytochemical withaferin A (WA), have, therefore, gained pharmaceutical interest. Remarkably, WA is more effective than IBR in killing BTK-overexpressing glucocorticoid (GC)-resistant MM1R cells. To further characterize the kinase inhibitor profiles of WA and IBR in GC-resistant MM cells, we applied phosphopeptidome- and transcriptome-specific tyrosine kinome profiling. In contrast to IBR, WA was found to reverse BTK overexpression in GC-resistant MM1R cells. Furthermore, WA-induced cell death involves covalent cysteine targeting of Hinge-6 domain type tyrosine kinases of the kinase cysteinome classification, including inhibition of the hyperactivated BTK. Covalent interaction between WA and BTK could further be confirmed by biotin-based affinity purification and confocal microscopy. Similarly, molecular modeling suggests WA preferably targets conserved cysteines in the Hinge-6 region of the kinase cysteinome classification, favoring inhibition of multiple B-cell receptors (BCR) family kinases. Altogether, we show that WA's promiscuous inhibition of multiple BTK family tyrosine kinases represents a highly effective strategy to overcome GC-therapy resistance in MM.

The steroidal lactone withaferin A (WFA) is a dietary phytochemical, derived from Withania somnifera. It exhibits a wide range of biological properties, including immunomodulatory, anti-inflammatory, antistress, and anticancer activities. Here we investigated the effect of WFA on T-cell motility, which is crucial for adaptive immune responses as well as autoimmune reactions. We found that WFA dose-dependently (within the concentration range of 0.3-1.25 μM) inhibited the ability of human T-cells to migrate via cross-linking of the lymphocyte function-associated antigen-1 (LFA-1) integrin with its ligand, intercellular adhesion molecule 1 (ICAM-1). Coimmunoprecipitation of WFA interacting proteins and subsequent tandem mass spectrometry identified a WFA-interactome consisting of 273 proteins in motile T-cells. In particular, our data revealed significant enrichment of the zeta-chain-associated protein kinase 70 (ZAP70) and cytoskeletal actin protein interaction networks upon stimulation. Phospho-peptide mapping and kinome analysis substantiated kinase signaling downstream of ZAP70 as a key WFA target, which was further confirmed by bait-pulldown and Western immunoblotting assays. The WFA-ZAP70 interaction was disrupted by a disulfide reducing agent dithiothreitol, suggesting an involvement of cysteine covalent binding interface. In silico docking predicted WFA binding to ZAP70 at cystine 560 and 564 residues. These findings provide a mechanistic insight whereby WFA binds to and inhibits the ZAP70 kinase and impedes T-cell motility. We therefore conclude that WFA may be exploited to pharmacologically control host immune responses and potentially prevent autoimmune-mediated pathologies. Ministry of Education (MOE) National Medical Research Council (NMRC) National Research Foundation (NRF) Published version This research was supported, in part, by the Singapore Ministry of Education (MOE) under its MOE Academic Research Fund (AcRF) Tier 2 Grant (MOE2017-T2-2-004), MOEAcRF Tier 1 Grant (2020-T1-001-062) and the National Research Foundation Singapore under its Open Fund–Large Collaborative Grant (OFLCG18May-0028) and administered by the Singapore Ministry of Health’s National Medical Research Council (NMRC). W. V. B. acknowledges funding support from the Foundation Against Cancer (Grant number-7872, Belgium), Hercules Foundation (Grant numberAUHA/13/012, Belgium), and Research Foundation - Flanders (FWO, grant numbers G059713N/G079614N, Belgium).

Delivery of conventional antisense oligonucleotides or small interfering RNA (siRNA) molecules into hematolymphoid cells for targeted gene silencing has been proven to be difficult. Here, we describe a simple protocol to knockdown specific gene(s) in malignant hematolymphoid cells using "GapmeR." This protocol could be applicable to a wide range of cell-types and thus solves an important problem for researchers working with cell lines or primary cells derived from patients with hematolymphoid malignancies.

Bacterial colonization of acute and chronic wounds is often associated with delayed wound healing and prolonged hospitalization. The rise of multi-drug resistant bacteria and the poor biocompatibility of topical antimicrobials warrant safe and effective antimicrobials. Antimicrobial agents that target microbial membranes without interfering with the mammalian cell proliferation and migration hold great promise in the treatment of traumatic wounds. This article reports the utility of superhydrophilic electrospun gelatin nanofiber dressings (NFDs) containing a broad-spectrum antimicrobial polymer, e-polylysine (ePL), crosslinked by polydopamine (pDA) for treating second-degree burns. In a porcine model of partial thickness burns, NFDs promoted wound closure and reduced hypertrophic scarring compared to untreated burns. Analysis of NFDs in contact with the burns indicated that the dressings trap early colonizers and elicit bactericidal activity, thus creating a sterile wound bed for fibroblasts migration and re-epithelialization. In support of these observations, in porcine models of Pseudomonas aeruginosa and Staphylococcus aureus colonized partial thickness burns, NFDs decreased bacterial bioburden and promoted wound closure and re-epithelialization. NFDs displayed superior clinical outcome than standard-of-care silver dressings. The excellent biocompatibility and antimicrobial efficacy of the newly developed dressings in pre-clinical models demonstrate its potential for clinical use to manage infected wounds without compromising tissue regeneration.

Delivery of conventional antisense oligonucleotides or small interfering RNA (siRNA) molecules into hematolymphoid cells for targeted gene silencing has been proven to be difficult. Here, we describe a simple protocol to knockdown specific gene(s) in malignant hematolymphoid cells using "GapmeR." This protocol could be applicable to a wide range of cell-types and thus solves an important problem for researchers working with cell lines or primary cells derived from patients with hematolymphoid malignancies.

Catheter associated infections (CAIs) are the major cause of nosocomial infections leading to increased morbidity, mortality rates and economical loss. Though the antibiotic coated surface modified catheters are reported to be effective in preventing CAIs, presence of sub-lethal concentrations of antibiotics in long term instilled catheters poses a risk of development and spread of drug resistant microbial strains. Herein, we have developed an antibiotic-free alternative strategy to coat catheter surfaces using pyrogallol (PG) and metal ions (Ag+/Mg2+). Surface characteristics, antimicrobial and anti-biofilm properties with hemocompatibility of the coated catheters were studied. Structural characteristics of coated catheters were similar to the uncoated catheters with improved wettability. All the coated catheters with PG and different PG/metal ion combinations exhibited broad spectrum antibacterial activity. Catheters coated with PG/metal ions combination showed effective antibiofilm properties against MRSA strains. None of the coated catheters showed any significant hemolysis for rabbit erythrocytes. In addition, polypyrogallol (pPG) coating attenuated the hemolytic properties of silver without altering the antimicrobial properties. The inherent antimicrobial properties of the coating agent along with antimicrobial metal ions broaden the application landscape which includes coating of other medical devices, clean room construction and development of antimicrobial surfaces. The chemical formulation can also be used to design antiseptic solutions to prevent healthcare associated infections. NRF (Natl Research Foundation, S’pore) NMRC (Natl Medical Research Council, S’pore) MOH (Min. of Health, S’pore) Accepted version

David Hutchinson,1,2 Judith Müller,3 Joseph E McCarthy,4 Yurii K Gun’ko,4,5 Navin Kumar Verma,6 Xuezhi Bi,7 Luisana Di Cristo,8 Laura Kickham,8 Dania Movia,8 Adriele Prina-Mello,5,8 Yuri Volkov5,8,9 1Royal Cornwall Hospital NHS Trust, Treliske, 2University of Exeter Medical School Cornwall, UK; 3University of Basel, Basel, Switzerland; 4School of Chemistry, 5Advanced Materials for BioEngineering Research Centre (AMBER), Trinity College Dublin, Dublin, Ireland; 6Lee Kong Chian School of Medicine, Nanyang Technological University, 7Bioprocessing Technology Institute, A*STAR Graduate Academy, Singapore; 8Department of Clinical Medicine, School of Medicine, Trinity College Dublin, Dublin, Ireland; 9International Laboratory of Magnetically Controlled Nanosystems for Theranostics of Oncological and Cardiovascular Diseases, ITMO University, St. Petersburg, Russia Objective: The objective of the study was to determine whether the cadmium-derived materials induce intracellular protein citrullination. Methods: Human A549 lung epithelial cells were exposed to cadmium in soluble and nanoparticulate forms represented by cadmium chloride (CdCl2) and cadmium oxide (CdO), respectively, and their combinations with ultrafine carbon black (ufCB) produced by high temperature combustion, imitating cigarette burning. Protein citrullination in cell lysates was analyzed by Western immunoblotting and verified by immunofluorescent confocal microscopy. Target citrullinated proteins were identified by proteomic analysis. Results: CdO, ufCB and its combination with CdCl2 and CdO after high temperature combustion induced protein citrullination in cultured human lung epithelial cells, as detected by immunoblotting with anti-citrullinated protein antibody. Cytokeratins of type II (1, 2, 5, 6A, 6B and 77) and type I (9, 10) were identified as major intracellular citrullination targets. Immunofluorescent staining confirmed the localization of citrullinated proteins both in the cytoplasm and cell nuclei. Conclusion: Cadmium oxide nanoparticle exposure facilitated post-translational citrullination of proteins. Keywords: cadmium, COPD, nanoparticles, cytokeratins, citrullination, autoimmunity, proteomics

There is a growing demand for durable advanced wound dressings for the management of persistent infections after deep burn injuries. Herein, we demonstrated the preparation of durable antimicrobial nanofiber mats, by taking advantage of strong interfacial interactions between polyhydroxy antibiotics (with varying number of single bondOH groups) and gelatin and their in-situ crosslinking with polydopamine (pDA) using ammonium carbonate diffusion method. Polydopamine crosslinking did not interfere with the antimicrobial efficacy of the loaded antibiotics. Interestingly, incorporation of antibiotics containing more number of alcoholic single bondOH groups (NOH ≥ 5) delayed the release kinetics with complete retention of antimicrobial activity for an extended period of time (20 days). The antimicrobials-loaded mats displayed superior mechanical and thermal properties than gelatin or pDA-crosslinked gelatin mats. Mats containing polyhydroxy antifungals showed enhanced aqueous stability and retained nanofibrous morphology under aqueous environment for more than 4 weeks. This approach can be expanded to produce mats with broad spectrum antimicrobial properties by incorporating the combination of antibacterial and antifungal drugs. Direct electrospinning of vancomycin-loaded electrospun nanofibers onto a bandage gauze and subsequent crosslinking produced non-adherent durable advanced wound dressings that could be easily applied to the injured sites and readily detached after treatment. In a partial thickness burn injury model in piglets, the drug-loaded mats displayed comparable wound closure to commercially available silver-based dressings. This prototype wound dressing designed for easy handling and with long-lasting antimicrobial properties represents an effective option for treating life-threatening microbial infections due to thermal injuries. NRF (Natl Research Foundation, S’pore) MOE (Min. of Education, S’pore) NMRC (Natl Medical Research Council, S’pore) MOH (Min. of Health, S’pore) Published version

The direct tracking of cells using fluorescent dyes is a constant challenge in cell therapy due to aggregation-induced quenching (ACQ) effect and biocompatibility issues. Here, we demonstrate the development of a biocompatible and highly efficient aggregation-induced emission (AIE)-active pseudorotaxane luminogen based on tetraphenylethene conjugated poly(ethylene glycol) (TPE-PEG2) (guest) and α-cyclodextrin (α-CD) (host). It is capable of showing significant fluorescent emission enhancement at the 400-600 nm range when excited at 388 nm, without increasing the concentration of AIE compound. The fluorescent intensity of TPE-PEG2 solution was effectively enhanced by 4-12 times with gradual addition of 1-4 mM of α-CD. 2D NOSEY 1H NMR revealed clear correlation spots between the characteristic peaks of α-CD and PEG, indicating the interaction between protons of ethylene glycol and cyclodextrin, and the structures are mainly based on threaded α-CD. The host-guest complex exhibits boosted fluorescent emission because the PEG side chains are confined in "nano-cavities" (host), thus, applying additional restriction on intermolecular rotation of TPE segments. In vitro cell experiments demonstrated the potential of AIE-active pseudorotaxane polymer as a biocompatible bioimaging probe. ASTAR (Agency for Sci., Tech. and Research, S’pore) MOE (Min. of Education, S’pore) Accepted version

The immune system and its components defend our body against diverse pathogens and help in maintaining tissue homeostasis. Immune cells are highly dynamic in terms of their growth, migration, differentiation, and effector functions, and adopt diverse metabolic configurations to respond to varying immunological challenges. Growing body of evidence suggests that metabolic pathways fuel immune cells for their functioning, including T-cell migration to the site of infection. This chapter provides detailed methodology for the efficient extraction of T-cell metabolites for successful downstream immunometabolomic profiling of motile T-lymphocytes.

The ability of activated T-lymphocytes to transmigrate toward certain chemokine is one of their characteristic functional properties. Here, we provide step-wise details about an in vitro technique to quantify the kinetics of chemotactic behavior of LFA-1-stimulated T-lymphocytes. The method described herein utilizes a noninvasive electrical impedance-based detection system to monitor T-cell chemotaxis in real-time.

The immune synapse is a complex cellular structure that enables cell-cell communications between immune cells, mainly at the interface of an effector T-cell and an antigen-presenting cell (APC) that expresses the appropriate peptide-MHC complexes. With progressive technological advances, there has been increasing interest in understanding molecular events that take place in motile T-lymphocytes at the immune synapse. Here, we provide an optimized method to induce the formation of an immune synapse between a T-cell and an APC in vitro. The experimental protocol described herein would be useful in characterizing the role of cell surface molecules and downstream signaling pathways in the context of cell-to-cell communications between T-cells and APCs.

Visualization of signal transduction events in T-cells has always been a challenge due to their miniscule size. Recent advancement in super-resolution microscopy techniques presents many new opportunities to navigate the spatial and temporal signaling cross-talks in motile T-cells. Here, we provide technical details, optimal conditions, and critical practical considerations that need to be taken into account during cell handling, sample preparation, and image acquisition of motile T-cells for performing three-dimensional structured illumination microscopy (3D-SIM).