Heat-killed probiotic Levilactobacillus brevis MKAK9 and its exopolysaccharide promote longevity by modulating aging hallmarks and enhancing immune responses in Caenorhabditis elegans.

BACKGROUND: Proteostasis is a critical aging hallmark responsible for removing damaged or misfolded proteins and their aggregates by improving proteasomal degradation through the autophagy-lysosome pathway (ALP) and the ubiquitin-proteasome system (UPS). Research on the impact of heat-killed probiotic bacteria and their structural components on aging hallmarks and innate immune responses is scarce, yet enhancing these effects could potentially delay age-related diseases. RESULTS: This study introduces a novel heat-killed Levilactobacillus brevis strain MKAK9 (HK MKAK9), along with its exopolysaccharide (EPS), demonstrating their ability to extend longevity by improving proteostasis and immune responses in wild-type Caenorhabditis elegans. We elucidate the underlying mechanisms through a comprehensive approach involving mRNA- and small RNA sequencing, proteomic analysis, lifespan assays on loss-of-function mutants, and quantitative RT-PCR. Mechanistically, HK MKAK9 and its EPS resulted in downregulation of the insulin-like signaling pathway in a DAF-16-dependent manner, enhancing protein ubiquitination and subsequent proteasomal degradation through activation of the ALP pathway, which is partially mediated by microRNA mir-243. Importantly, autophagosomes engulf ubiquitinylated proteins, as evidenced by increased expression of the autophagy receptor sqst-3, and subsequently fuse with lysosomes, facilitated by increased levels of the lysosome-associated membrane protein (LAMP) lmp-1, suggesting the formation of autolysosomes for degradation of the selected cargo. Moreover, HK MKAK9 and its EPS activated the p38 MAPK pathway and its downstream SKN-1 transcription factor, which are known to regulate genes involved in innate immune response (thn-1, ilys-1, cnc-2, spp-9, spp-21, clec-47, and clec-266) and antioxidation (sod-3 and gst-44), thereby reducing the accumulation of reactive oxygen species (ROS) at both cellular and mitochondrial levels. Notably, SOD-3 emerged as a transcriptional target of both DAF-16 and SKN-1 transcription factors. CONCLUSION: Our research sets a benchmark for future investigations by demonstrating that heat-killed probiotic and its specific cellular component, EPS, can downregulate the insulin-signaling pathway, potentially improving the autophagy-lysosome pathway (ALP) for degrading ubiquitinylated proteins and promoting organismal longevity. Additionally, we discovered that increased expression of microRNA mir-243 regulates insulin-like signaling and its downstream ALP pathway. Our findings also indicate that postbiotic treatment may bolster antioxidative and innate immune responses, offering a promising avenue for interventions in aging-related diseases.

Advances in ex vivo expansion of hematopoietic stem and progenitor cells for clinical applications.

As caretakers of the hematopoietic system, hematopoietic stem cells assure a lifelong supply of differentiated populations that are responsible for critical bodily functions, including oxygen transport, immunological protection and coagulation. Due to the far-reaching influence of the hematopoietic system, hematological disorders typically have a significant impact on the lives of individuals, even becoming fatal. Hematopoietic cell transplantation was the first effective therapeutic avenue to treat such hematological diseases. Since then, key use and manipulation of hematopoietic stem cells for treatments has been aspired to fully take advantage of such an important cell population. Limited knowledge on hematopoietic stem cell behavior has motivated in-depth research into their biology. Efforts were able to uncover their native environment and characteristics during development and adult stages. Several signaling pathways at a cellular level have been mapped, providing insight into their machinery. Important dynamics of hematopoietic stem cell maintenance were begun to be understood with improved comprehension of their metabolism and progressive aging. These advances have provided a solid platform for the development of innovative strategies for the manipulation of hematopoietic stem cells. Specifically, expansion of the hematopoietic stem cell pool has triggered immense interest, gaining momentum. A wide range of approaches have sprouted, leading to a variety of expansion systems, from simpler small molecule-based strategies to complex biomimetic scaffolds. The recent approval of Omisirge, the first expanded hematopoietic stem and progenitor cell product, whose expansion platform is one of the earliest, is predictive of further successes that might arise soon. In order to guarantee the quality of these ex vivo manipulated cells, robust assays that measure cell function or potency need to be developed. Whether targeting hematopoietic engraftment, immunological differentiation potential or malignancy clearance, hematopoietic stem cells and their derivatives need efficient scaling of their therapeutic potency. In this review, we comprehensively view hematopoietic stem cells as therapeutic assets, going from fundamental to translational.

Dual production of human mesenchymal stromal cells and derived extracellular vesicles in a dissolvable microcarrier-based stirred culture system.

BACKGROUND & AIMS: Cell therapies based on mesenchymal stromal cells (MSCs) have gained an increasing therapeutic interest in the context of multiple disorders. Nonetheless, this field still faces important challenges, particularly concerning suitable manufacturing platforms. Here, we aimed at establishing a scalable culture system to expand umbilical cord-derived Wharton's jelly MSC (MSC(WJ)) and their derived extracellular vesicles (EVs) by using dissolvable microcarriers combined with xeno(geneic)-free culture medium. METHODS: MSC(WJ) isolated from three donors were cultured at a starting density of 1 × 106 cells per spinner flask, i.e., 2.8 × 103 cells per cm2 of dissolvable microcarrier surface area. After a 6-day expansion period of MSC(WJ), extracellular vesicles (EVs) were produced for 24 h. RESULTS: Taking advantage of an intermittent agitation regimen, we observed high adhesion rates to the microcarriers (over 90% at 24 h) and achieved 15.8 ± 0.7-fold expansion after 6 days of culture. Notably, dissolution of the microcarriers was achieved through a pectinase-based solution to recover the cell product, reducing the hurdles of downstream processing. MSC identity was validated by detecting the characteristic MSC immunophenotype and by multilineage differentiation assays. Considering the growing interest in MSC-derived EVs, which are known to be mediators of the therapeutic features of MSC, this platform also was evaluated for EV production. Upon a 24-h period of conditioning, secreted EVs were isolated by ultrafiltration followed by anion-exchange chromatography and exhibited the typical cup-shaped morphology, small size distribution (162.6 ± 30.2 nm) and expressed EV markers (CD63, CD9 and syntenin-1). CONCLUSIONS: Taken together, we established a time-effective and robust scalable platform that complies with clinical-grade standards for the dual production of MSC(WJ) and their derived EV.

A passage-free, simplified, and scalable novel method for iPSC generation in three-dimensional culture.

Induced pluripotent stem cells (iPSCs) have immense potential for use in disease modeling, etiological studies, and drug discovery. However, the current workflow for iPSC generation and maintenance poses challenges particularly during the establishment phase when specialized skills are required. Although three-dimensional culture systems offer scalability for maintaining established iPSCs, the enzymatic dissociation step is complex and time-consuming. In this study, a novel approach was developed to address these challenges by enabling iPSC generation, maintenance, and differentiation without the need for two-dimensional culture or enzymatic dissociation. This streamlined method offers a more convenient workflow, reduces variability and labor for technicians, and opens up avenues for advancements in iPSC research and broader applications.

Memory Guided Transformer With Spatio-Semantic Visual Extractor for Medical Report Generation.

Medicalimaging-based report writing for effective diagnosis in radiology is time-consuming and can be error-prone by inexperienced radiologists. Automatic reporting helps radiologists avoid missed diagnoses and saves valuable time. Recently, transformer-based medical report generation has become prominent in capturing long-term dependencies of sequential data with its attention mechanism. Nevertheless, input features obtained from traditional visual extractor of conventional transformers do not capture spatial and semantic information of an image. So, the transformer is unable to capture fine-grained details and may not produce detailed descriptive reports of radiology images. Therefore, we propose a spatio-semantic visual extractor (SSVE) to capture multi-scale spatial and semantic information from radiology images. Here, we incorporate two types of networks in ResNet 101 backbone architecture, i.e. (i) deformable network at the intermediate layer of ResNet 101 that utilizes deformable convolutions in order to obtain spatially invariant features, and (ii) semantic network at the final layer of backbone architecture which uses dilated convolutions to extract rich multi-scale semantic information. Further, these network representations are fused to encode fine-grained details of radiology images. The performance of our proposed model outperforms existing works on two radiology report datasets, i.e., IU X-ray and MIMIC-CXR.

Leveraging Lymphatic System Targeting in Systemic Lupus Erythematosus for Improved Clinical Outcomes.

The role of advanced drug delivery strategies in drug repositioning and minimizing drug attrition rates, when applied early in drug discovery, is poised to increase the translational impact of various therapeutic strategies in disease prevention and treatment. In this context, drug delivery to the lymphatic system is gaining prominence not only to improve the systemic bioavailability of various pharmaceutical drugs but also to target certain specific diseases associated with the lymphatic system. Although the role of the lymphatic system in lupus is known, very little is done to target drugs to yield improved clinical benefits. In this review, we discuss recent advances in drug delivery strategies to treat lupus, the various routes of drug administration leading to improved lymph node bioavailability, and the available technologies applied in other areas that can be adapted to lupus treatment. Moreover, this review also presents some recent findings that demonstrate the promise of lymphatic targeting in a preclinical setting, offering renewed hope for certain pharmaceutical drugs that are limited by efficacy in their conventional dosage forms. These findings underscore the potential and feasibility of such lymphatic drug-targeting approaches to enhance therapeutic efficacy in lupus and minimize off-target effects of the pharmaceutical drugs. SIGNIFICANCE STATEMENT: The World Health Organization estimates that there are currently 5 million humans living with some form of lupus. With limited success in lupus drug discovery, turning to effective delivery strategies with existing drug molecules, as well as those in the early stage of discovery, could lead to better clinical outcomes. After all, effective delivery strategies have been proven to improve treatment outcomes.

Scope and challenges of seaweed utilization in food and nutraceutical industry in India: a review.

Seaweeds are an excellent source of unique antioxidant phytochemicals, dietary fibres, essential amino acids, vitamins, polyunsaturated fatty acids and minerals. The presence of such structurally diverse and high value bioactive compounds has led to popularization of seaweed as functional food ingredient in global health supplement market. India, with a long coastline of 8100 km and exclusive economic zone of 2.17 million km2, is rich in diverse seaweed resources belonging to almost 700 species. However, food and nutraceutical application of Indian seaweed is highly constrained. Apart from Kappaphycus alvarezii, there is no systematic commercial cultivation of seaweed in India. The regulatory framework for use of seaweed as food is still developing and consumer acceptance is still low. However, there is a timely and renewed interest from different government agencies and research organisations to develop a thriving food and nutraceutical industry using India's vast seaweed resources. The review briefly describes the nutritional and functional food potential of the seaweed and goes on to discuss the scope of seaweed utilization in food and nutraceutical industry in India. Further, the review has identified the regulatory challenges and quality control requirements for use of seaweeds in food and nutraceuticals.

Identification of a Staphylococcus aureus amidase catalytic domain inhibitor to prevent biofilm formation by sequential virtual screening, molecular dynamics simulation and biological evaluation.

Staphylococcus aureus (S. aureus) is one of the common causes of implant associated biofilm infections and their biofilms are resistant to antibiotics. S. aureus amidase (AM) protein, a cell wall hydrolase that cleaves the amide bond between N-acetylmuramic acid and L-alanine residue of the stem peptide, is several fold over-expressed under biofilm conditions. Previous studies demonstrated an autolysin mutant in S. aureus that lacks the AM protein, is highly impaired in biofilm development. We carried out a structure-based small molecule design using the crystal structure of AM protein catalytic domain to identify inhibitors that can block amidase activity and therefore inhibits S. aureus biofilm formation. Sequential virtual screening followed by pharmacokinetic analysis and bioassay studies filtered 25 small molecules from different databases. Two compounds from the SPECS database, SPECS-1 and SPECS-2, were selected based on the best docking score and minimum biofilm inhibitory concentration towards S. aureus biofilms. SPECS-1 and SPECS-2 were further tested for their structural/energetic stability in complex with the AM protein using molecular dynamics simulation and MM-GBSA techniques. In vitro, biofilm inhibition studies on different surfaces confirmed that treatment with SPECS-1 and SPECS-2 at a concentration of 250 µg/ml exhibited significant prevention and disruption of S. aureus biofilms. Finally, the in vitro anti-biofilm activities of these two compounds were validated against Methicillin-resistant S. aureus clinical isolates. We concluded that the discovered compounds SPECS-1 and SPECS-2 are safe and exhibit biofilm preventive and disruption activity for inhibiting the S. aureus biofilms and hence can be used to treat implant-associated biofilm infections.

Analogs of FTY720 inhibit TRPM7 but not S1PRs and exert multimodal anti-inflammatory effects.

TRPM7, a TRP channel with ion conductance and kinase activities, has emerged as an attractive drug target for immunomodulation. Reverse genetics and cell biological studies have already established a key role for TRPM7 in the inflammatory activation of macrophages. Advancing TRPM7 as a viable molecular target for immunomodulation requires selective TRPM7 inhibitors with in vivo tolerability and efficacy. Such inhibitors have the potential to interdict inflammatory cascades mediated by systemic and tissue-specialized macrophages. FTY720, an FDA-approved drug for multiple sclerosis inhibits TRPM7. However, FTY720 is a prodrug and its metabolite, FTY720-phosphate, is a potent agonist of sphingosine-1-phosphate (S1P) receptors. In this study, we test non-phosphorylatable FTY720 analogs, which are inert against S1PRs and well tolerated in vivo, for activity against TRPM7 and tissue bioavailability. Using patch clamp electrophysiology, we show that VPC01091.4 and AAL-149 block TRPM7 current at low micromolar concentrations. In culture, they act directly on macrophages to blunt LPS-induced inflammatory cytokine expression, though this likely occurrs through multiple molecular targets. We found that VPC01091.4 has significant and rapid accumulation in the brain and lungs, along with direct anti-inflammatory action on alveolar macrophages and microglia. Finally, using a mouse model of endotoxemia, we show VPC01091.4 to be an efficacious anti-inflammatory agent that arrests systemic inflammation in vivo. Together, these findings identify novel small molecule inhibitors that allow TRPM7 channel inhibition independent of S1P receptor targeting which demonstrate potent, polymodal anti-inflammatory activities ex vivo and in vivo.

Recent Advances in mmWave-Radar-Based Sensing, Its Applications, and Machine Learning Techniques: A Review.

Human gesture detection, obstacle detection, collision avoidance, parking aids, automotive driving, medical, meteorological, industrial, agriculture, defense, space, and other relevant fields have all benefited from recent advancements in mmWave radar sensor technology. A mmWave radar has several advantages that set it apart from other types of sensors. A mmWave radar can operate in bright, dazzling, or no-light conditions. A mmWave radar has better antenna miniaturization than other traditional radars, and it has better range resolution. However, as more data sets have been made available, there has been a significant increase in the potential for incorporating radar data into different machine learning methods for various applications. This review focuses on key performance metrics in mmWave-radar-based sensing, detailed applications, and machine learning techniques used with mmWave radar for a variety of tasks. This article starts out with a discussion of the various working bands of mmWave radars, then moves on to various types of mmWave radars and their key specifications, mmWave radar data interpretation, vast applications in various domains, and, in the end, a discussion of machine learning algorithms applied with radar data for various applications. Our review serves as a practical reference for beginners developing mmWave-radar-based applications by utilizing machine learning techniques.

Novel TRPM7 inhibitors with potent anti-inflammatory effects in vivo.

TRPM7, a TRP channel with ion conductance and kinase activities, has emerged as an attractive drug target for immunomodulation. Reverse genetics and cell biological studies have already established a key role for TRPM7 in the inflammatory activation of macrophages. Advancing TRPM7 as a viable molecular target for immunomodulation requires selective TRPM7 inhibitors with in vivo tolerability and efficacy. Such inhibitors have the potential to interdict inflammatory cascades mediated by systemic and tissue-specialized macrophages. FTY720, an FDA-approved drug for multiple sclerosis inhibits TRPM7. However, FTY720 is a prodrug and its metabolite, FTY720-phosphate, is a potent agonist of sphingosine 1-phosphate (S1P) receptors. In this study, we tested non-phosphorylatable FTY720 analogs, which are inert against S1PRs and well tolerated in vivo , for activity against TRPM7 and tissue bioavailability. Using patch clamp electrophysiology, we show that VPC01091.4 and AAL-149 block TRPM7 current at low micromolar concentrations. In culture, they act directly on macrophages to blunt LPS-induced inflammatory cytokine expression, an effect that is predominantly but not solely mediated by TRPM7. We found that VPC01091.4 has significant and rapid accumulation in the brain and lungs, along with direct anti-inflammatory action on alveolar macrophages and microglia. Finally, using a mouse model of endotoxemia, we show VPC01091.4 to be an efficacious anti-inflammatory agent that arrests systemic inflammation in vivo . Together, these findings identify novel small molecule inhibitors that allow TRPM7 channel inhibition independent of S1P receptor targeting. These inhibitors exhibit potent anti-inflammatory properties that are mediated by TRPM7 and likely other molecular targets that remain to be identified.

Probing the origins of programmed death ligand-1 inhibition by implementing machine learning-assisted sequential virtual screening techniques.

PD-L1 is a key immunotarget involved in binding to its receptor PD-1. PD-L1/PD-1 interface blocking using antibodies (or small molecules) is the central area of interest for tumor suppression in various cancers. Blocking the PD-L1/PD-1 pathway in the tumor cells results in its immune activation and destruction, and thereby restoring the T-cell proliferation and cytokine production. The active binding site interface residues of PD-L1/PD-1 were experimentally known and proven by structural biology and site-directed mutagenesis studies. Structure-based molecular design technique was employed to identify the inhibitors for blocking the PD-L1/PD-1 interface. Nine hits to leads were identified from the SPECS small molecule database by machine learning, molecular docking, and molecular dynamics simulation techniques. Following this, a machine learning-assisted QSAR modeling approach was implemented using ChEMBL database to gain insights into the inhibitory potential of PD-L1 inhibitors and predict the activity of our previously screened nine hit molecules. The best leads identified in the present study bind strongly with the active sites of PD-L1/PD-1 interface residues, which include A121, M115, I116, S117, I54, Y56, D122, and Y123. These computational leads are considered promising molecules for further in vitro and in vivo analysis to be developed as potential PD-L1 checkpoint inhibitors to cure different types of cancers.

MAPP Compatibilized Recycled Woodchips Reinforced Polypropylene Composites with Exceptionally High Strength and Stability.

Wood chips were used in their original form without any physical or chemical treatment as reinforcement for polypropylene to develop composites as potential replacement for medium density fiber (MDF) boards, gypsum based false ceiling and other building materials. Wood chips are generated as byproducts and have limited and low value applications. Composites with up to 90% wood chips were developed through compression molding and the mechanical, acoustic and thermal properties were studied. Further, maleated polypropylene (MAPP) was used (1-5% w/w based on woodchips used) as compatibilizer and changes in properties were recorded. Up to 300% increase in tensile properties were observed when 5% compatibilizer was present. Tensile properties of the composites containing MAPP were higher than that of commercially available medium density plywood boards and also gypsum based ceiling tiles. Addition of MAPP did not change thermal conductivity but decreased sound absorption. Wood chips reinforced PP composites containing MAPP show exceedingly high properties and could replace particle, fiber boards and other building materials in current use. Utilizing the wood waste also results in environmentally friendly, sustainable and low cost building materials.

The G-protein-coupled estrogen receptor, a gene co-expressed with ERα in breast tumors, is regulated by estrogen-ERα signalling in ERα positive breast cancer cells.

GPER is a seven transmembrane G-protein-coupled estrogen receptor that mediates rapid estrogen actions. Large volumes of data have revealed its association with clinicopathological variables in breast tumors, role in epidermal growth factor (EGF)-like effects of estrogen, potential as a therapeutic target or a prognostic marker, and involvement in endocrine resistance in the face of tamoxifen agonism. GPER cross-talks with estrogen receptor alpha (ERα) in cell culture models implicating its role in the physiology of normal or transformed mammary epithelial cells. However, discrepancies in the literature have obfuscated the nature of their relationship, its significance, and the underlying mechanism. The purpose of this study was to assess the relationship between GPER, and ERα in breast tumors, to understand the mechanistic basis, and to gauge its clinical significance. We mined The Cancer Genome Atlas (TCGA)-BRCA data to examine the relationship between GPER and ERα expression. GPER mRNA, and protein expression were analyzed in ERα-positive or -negative breast tumors from two independent cohorts using immunohistochemistry, western blotting, or RT-qPCR. The Kaplan-Meier Plotter (KM) was employed for survival analysis. The influence of estrogen in vivo was studied by examining GPER expression levels in estrus or diestrus mouse mammary tissues, and the impact of 17ß-estradiol (E2) administration in juvenile or adult mice. The effect of E2, or propylpyrazoletriol (PPT, an ERα agonist) stimulation on GPER expression was studied in MCF-7 and T47D cells, with or without tamoxifen or ERα knockdown. ERα-binding to the GPER locus was explored by analysing ChIP-seq data (ERP000380), in silico prediction of estrogen response elements, and chromatin immunoprecipitation (ChIP) assay. Clinical data revealed significant positive association between GPER and ERα expression in breast tumors. The median GPER expression in ERα-positive tumors was significantly higher than ERα-negative tumors. High GPER expression was significantly associated with longer overall survival (OS) of patients with ERα-positive tumors. In vivo experiments showed a positive effect of E2 on GPER expression. E2 induced GPER expression in MCF-7 and T47D cells; an effect mimicked by PPT. Tamoxifen or ERα-knockdown blocked the induction of GPER. Estrogen-mediated induction was associated with increased ERα occupancy in the upstream region of GPER. Furthermore, treatment with 17ß-estradiol or PPT significantly reduced the IC50 of the GPER agonist (G1)-mediated loss of MCF-7 or T47D cell viability. In conclusion, GPER is positively associated with ERα in breast tumors, and induced by estrogen-ERα signalling axis. Estrogen-mediated induction of GPER makes the cells more responsive to GPER ligands. More in-depth studies are warranted to establish the significance of GPER-ERα co-expression, and their interplay in breast tumor development, progression, and treatment.

A biosurfactant-producing novel bacterial strain isolated from vermicompost having multiple plant growth-promoting traits.

The nutrient-rich vermicompost which is used as manure for the growth and development of plants is rich in microbial flora. These microbes protect the plants against several infectious pathogenic microbes. As certain microbes are known to produce biosurfactants as metabolites, an investigation was carried out to isolate biosurfactant-producing bacterial strains from vermicompost with the efficient antifungal property. From the study, it was revealed that biosurfactant-producing bacterial strains are present in the vermicompost. A total of nine bacterial strains were isolated from the vermicompost. Among them, one most efficient biosurfactant-producing bacterial strains with antifungal properties have been screened. After molecular characterization of the isolated strain, it was revealed that the bacterial strain is Bacillus licheniformis strain SCV1. The strain produces 3.4 ± 0.1 g/L of crude biosurfactant, which when column purified yields 3.1 ± 0.1 g/L of biosurfactant. The biosurfactant exhibited excellent emulsifying activity (E24 ) of 96.56% against crude oil. The produced biosurfactant was identified as a lipopeptide consisting of a mixer of surfactin and iturin. Furthermore, the biosurfactant exhibited significant antifungal activity against a wide range of phytopathogens, showing 76.3% inhibition against Sclerotinia sclerotiorum, 53% inhibition against Colletotrichum gloeosporioides, 51% against Fusarium verticillioides, and 36% against Corynespora cassicolla. Along with antifungal activities, the stain was found to exhibit multiple plant growth-promoting traits. This study, thus indicates that vermicompost might contain biosurfactant-producing microbes which can render protection to the plant against various phytopathogens by the production of biosurfactants and can also stimulate plant growth.

Effects of different cooking methods on the proximate composition and physical properties of Brown shrimp (Metapenaeus dobsonii) during cooking and freezing cycle.

Present study aimed to evaluate the changes in proximate composition and physical attributes in brown shrimp (Metapenaeus dobsonii) exposed to different methods of cooking followed by freezing. For this, three different grades (100/200, 200/300, and 300/500 numbers per kg) of brown shrimp were cooked at 90°C till the core temperature of the product reaches 85°C using hot water, steam, and microwave (400W) techniques. The changes in yield, cooking loss, proximate composition, textural, and colour profile were assessed for cooked shrimps. The cooking loss was higher for larger grades of shrimp, whereas shrimp cooked using hot water exhibited the highest cooking loss. Lowest cooking loss was observed for microwave-cooked shrimp. Moisture content decreased after cooking whereas protein, fat, ash, and calorie content increased. After cooking, different grades of shrimp showed an increase in their lightness (L*), redness (a*), and yellowness (b*) values. The smaller grade shrimp exhibited lower value for cohesiveness, hardness, chewiness, and gumminess. Different cooking techniques yielded cooked shrimp of varying hardness values.

Identification of a PD1/PD-L1 inhibitor by structure-based pharmacophore modelling, virtual screening, molecular docking and biological evaluation.

PD-1/PD-L1 is a critical druggable target for immunotherapy against sepsis. Chemoinformatics techniques involved the structure-based 3D pharmacophore model development followed by virtual screening of small molecule databases to identify the small molecules against PD-L1 pathway inhibition. Raltitrexed and Safinamide act as potent repurposed drugs, and three other Specs database compounds using in silico methods. These compounds were screened based on the pharmacophore fit score and binding affinity towards the active site of the PD-L1 protein. In silico pharmacokinetic profiling of these screened compounds was done to test their biological activity. Next, experimental validation of the best four virtually screened hits was done in vitro for its hemocompatibility and cytotoxicity. Among these, Raltitrexed, Safinamide and Specs compound (AK-968/40642641) effectively increased the proliferation of immune cells and IFN-γ production. These compounds can act as potent PDL-1 inhibitors for adjuvant therapy against sepsis.

Comparison of the Aotearoa New Zealand Early Warning Score and National Early Warning Score to predict adverse inpatient events in a vital sign dataset.

Aotearoa New Zealand uses a single early warning score (EWS) across all public and private hospitals to detect adult inpatient physiological deterioration. This combines the aggregate weighted scoring of the UK National Early Warning Score with single parameter activation from Australian medical emergency team systems. We conducted a retrospective analysis of a large vital sign dataset to validate the predictive performance of the New Zealand EWS in discriminating between patients at risk of serious adverse events and compared this with the UK EWS. We also compared predictive performance for patients admitted under medical vs. surgical specialties. A total of 1,738,787 aggregate scores (13,910,296 individual vital signs) were obtained from 102,394 hospital admissions to six hospitals within the Canterbury District Health Board of New Zealand's South Island. Predictive performance of each scoring system was determined using area under the receiver operating characteristic curve. Analysis showed that the New Zealand EWS is equivalent to the UK EWS in predicting patients at risk of serious adverse events (cardiac arrest, death and/or unanticipated ICU admission). Area under the receiver operating characteristic curve for both EWSs for any adverse outcome was 0.874 (95%CI 0.871-0.878) and 0.874 (95%CI 0.870-0.877), respectively. Both EWSs showed superior predictive value for cardiac arrest and/or death in patients admitted under surgical rather than medical specialties. Our study is the first validation of the New Zealand EWS in predicting serious adverse events in a broad dataset and supports previous work showing the UK EWS has superior predictive performance in surgical rather than medical patients.

Natural killer cells suppress human cutaneous squamous cell carcinoma cancer cell survival and tumor growth.

Cutaneous squamous cell carcinoma (CSCC), which develops in response to ultraviolet irradiation exposure, is among the most common cancers. CSCC lesions can be removed by surgical excision, but 4.5% of these cancers reappear as aggressive and therapy-resistant tumors. CSCC tumors display a high mutation burden, and tumor frequency is dramatically increased in immune-suppressed patients, indicating a vital role for the immune system in controlling cancer development. Natural killer cells (NK cells) play a key role in cancer immune surveillance, and recent studies suggest that NK cells from healthy donors can be expanded from peripheral blood for use in therapy. In the present study, we test the ability of ex vivo expanded human NK cells to suppress the CSCC cell cancer phenotype and reduce tumor growth. We expanded human NK cells from multiple healthy donors, in the presence of IL-2, and tested their ability to suppress the CSCC cell cancer phenotype. NK cell treatment produced a dose-dependent reduction in SCC-13 and HaCaT cell spheroid growth and matrigel invasion and induced SCC-13 and HaCaT cell apoptosis as evidenced by increased procaspase 9, procaspase 3, and PARP cleavage. Moreover, two important CSCC cell pro-cancer signaling pathways, YAP1/TAZ/TEAD and MEK1/2-ERK1/2, were markedly reduced. Furthermore, tail-vein injection of NK cells markedly suppressed the growth of SCC-13 xenograft tumors in NSG mice, which was also associated with a reduction in YAP1 and MEK1/2-P levels and enhanced apoptosis. These findings show that NK cell treatment suppresses CSCC cell spheroid formation, invasion, viability, and tumor growth, suggesting NK cell treatment may be a candidate therapy for CSCC.