Cardiovascular diseases are the leading cause of death globally. Myocardial infarction in particular leads to a high rate of mortality, and in the case of survival, to a loss of myocardial functionality due to post-infarction necrosis. This functionality can be restored by cell therapy or biomaterial implantation, and the need for a rapid regeneration has led to the development of bioactive patches, in particular through the incorporation of growth factors (GF). In this work, we designed hybrid patches composed of polymer nanofibers loaded with HGF and IGF and associated with a collagen membrane. Among the different copolymers studied, the polymers and their porogens PLA-Pluronic-PLA + PEG and PCL + Pluronic were selected to encapsulate HGF and IGF. While 89 and 92% of IGF were released in 2 days, HGF was released up to 58% and 50% in 35 days from PLA-Pluronic-PLA + PEG and PCL + Pluronic nanofibers, respectively. We also compared two ways of association for the loaded nanofibers and the collagen membrane, namely a direct deposition of the nanofibers on a moisturized collagen membrane (wet association), or entrapment between collagen layers (sandwich association). The interfacial cohesion and the degradation properties of the patches were evaluated. We also show that the sandwich association decreases the burst release of HGF while increasing the release efficiency. Finally, we show that the patches are cytocompatible and that the presence of collagen and IGF promotes the proliferation of C2C12 myoblast cells for 11 days. Taken together, these results show that these hybrid patches are of interest for cardiac muscle regeneration. Cardiovascular disease is the leading cause of death worldwide. Myocardial infarction and coronary heart diseases were responsible for 7.4 million deaths in 2015, according to the World Health Organisation . In contIn addition, to promote the integration of the nanofibers into the tissue, it has been shown that the presence of collagen, naturally present in cardiac tissue, is efficient . While rIn this study, we designed and prepared hybrid cardiac patches composed of IGF- and HGF-loaded degradable polymeric nanofibers associated with a collagen membrane. For this purpose, we synthesized different (co)polymers based on PLA and PCL in order to modulate the release kinetics of GFs. The nanofibers were then associated by different methods with a collagen membrane, and the impact of the association on GF release was evaluated. Finally, we studied the cytocompatibility and bioactivity of the nanofiber patch, demonstrating the interest of such a patch for cardiac regeneration.2), phosphate buffer solution (PBS), Bovine serum albumin, heat shock fraction V (BSA), Pluronic® F-127 , Sodium hydroxide (40 g·mol−1), Polycaprolactone (PCL) , and dichloromethane (DCM)  were supplied by Sigma. L-lactide (L-LA), D,L-lactide , and glycolide were purchased from Purac. Collagen membrane (CCC sterilized membrane) and collagen solution were kindly supplied by Viscofan Bioengineering. 2,2,2-trifluoroéthanol, 99+% (TFE) was purchased from Fisher Scientific and ethanol from Honeywell. Poly(ethylene glycol) (PEG) 10,000 g·mol−1 (ROTIPURAN® Ph.Eur) was received from Roth Sochiel EURL. Pierce™ BCA Protein Assay Kit (23227) and BSA FITC were purchased from Thermo Fischer Scientific, ELISA kit from Anticorps-enligne.fr, and growth factors  from Immunotools. PrestoBlueTM, Dulbecco’s Modified Eagle Medium (DMEM/F-12), Fetal Bovine Serum (FBS), penicillin, streptomycin, and glutamine were purchased from Invitrogen. BD Falcon™ Tissue Culture Polystyrene (TCPS) 24-well plates were purchased from Becton Dickinson and Viton® O-rings from Radiospares.D,L-lactide was purchased from Purac. Tin(II) 2-ethylhexanoate (Sn(Oct)−1  were introduce into a flask. For PLA50, benzyl alcohol was used as the initiator. After vacuum-nitrogen purge cycles, the flask was sealed under vacuum and placed at 125 °C for 8 days under stirring. The polymers were solubilized in DCM and purified by precipitation in cold ethanol. Finally, the resulting precipitate was dried under vacuum. The polymers were obtained with an average yield of 92%. The polymers’ molecular weights were calculated from the 1H NMR spectra using the integration of the peaks corresponding to the protons of the initiator and the methyne proton of lactic unit.PLA-Pluronic-PLA and PLGA-Pluronic-PLGA block copolymers were synthesized by ring opening polymerization following an already described procedure ,17,18. T−1 . Predete1H-NMR  δ (ppm) = 5.1 ; 4.3 ; 3.6 , 3.5 -CH2-O), 3.4 -CH2-O), 1.5 ), 1.1 -CH2-O).PLA-Pluronic-PLA: 1H-NMR  δ (ppm) = 5.1 , 4.8 , 3.6 , 3.5 -CH2-O), 3.4 -CH2-O), 1.5 ), 1.1 -CH2-O).PLGA-Pluronic-PLGA polymers: Number average molecular weight (Mn) and dispersity (Ð) of the polymers were determined by size exclusion chromatography (SEC) conducted on a Shimadzu LC-200 AD Prominence system, equipped with an RID-20A refractive index signal detector, a PLgel MIXED-C guard column , and two PLgel MIXED-C columns . Polymers were dissolved in THF (5 mg/mL), and solutions were filtered through a Millipore membrane before the injection of 100 μL into the system. All measurements were performed at 30 °C, and the mobile phase was THF at 1 mL/min flow and 30 °C. The number average molecular weight (Mn) and weight average molecular weight (Mw) were expressed according to a polystyrene calibration.1H NMR measurements were performed at 300 MHz with an AMX300 Bruker spectrometer using deuterated chloroform as the solvent. The chemical signals are expressed in ppm with respect to the tetramethylsilane (TMS) signal used as an internal reference.The w/w) and 29% (w/w), respectively. Then, 7 mg of BSA was loaded into 200 µL of polymer/porogen solution before being vigorously mixed and electrospun.A first series of BSA-loaded fibers was prepared for the initial screening and selection of polymers. Briefly, polymers were dissolved in TFE at different concentrations  and porov/v), and the resulting solution was mixed vigorously before being electrospun.GF-loaded nanofibers were prepared with the two selected polymers (PCL + Pluronic and PLA-Pluronic-PLA + PEG) . First, −1 for PCL + Pluronic and PLA-Pluronic-PLA + PEG, respectively. The protein-loaded nanofibers were collected on a flat aluminum foil of 5.2 × 5.2 cm, maintained at 15 cm of the needle. The electrospun mat was further cut into scaffolds of 1.3 × 1.3 cm that were dried vacuum overnight for complete elimination of the solvent.The electrospinning process was carried out at room temperature at a relative humidity of 20–40%. All the electrospinning parameters are detailed in https://imagej.nih.gov/ij/download.html, accessed on 20 July 2022) by randomly selecting 60 fibers.The surface morphology of the electrospun nanofibers was analyzed with SEM (Phenom ProX) after gold sputter coating. The average diameter of the fibers was measured using Image J software (BSA-loaded and GF-loaded nanofiber scaffolds weighing between 1 and 3 mg (1.3 × 1.3 cm) were incubated at 37 °C in 2 mL of PBS under shaking (100 rpm). At each time point, 1 mL of buffer was collected and replaced by an equal volume of fresh PBS. The amount of released BSA was determined by a Pierce BCA kit, following the manufacturer’s protocol and measuring the absorbance at 562 nm using a UV–vis spectrophotometer. The percentage of cumulated release of BSA was presented as mean ± standards deviation of three samples. The amount of growth factor was determined by an ELISA kit, following the manufacturer’s protocol and measuring absorbance at 450 nm.The experimental loading efficiency of BSA and GF was adapted from other protocols ,20 that Two methods were developed to associate collagen membranes and loaded nanofiber scaffolds. The first method, referred as the “sandwich association” method, associates the collagen membrane with the nanofibers thanks to a collagen solution. Briefly, 100 µL of collagen solution was deposited on 1.5 cm × 1.5 cm collagen membranes. Subsequently, the nanofibrous scaffold (1.3 cm × 1.3 cm) was placed in contact with the collagen solution before 100 µL was deposited again. The sandwich association was then freeze-dried overnight. The second method, referred as the “wet association” method, combines a collagen membrane (1.5 cm × 1.5 cm) humidified with water and a nanofiber scaffold (1.3 cm × 1.3 cm) placed directly on the moisturized collagen surface. After association, the construct was placed at 37 °C overnight to reinforce the interfacial interactions between the two layers.To quantify the interaction between the nanofiber scaffold and the collagen membrane, lap-shear type experiments were performed using an Instron 2710-203 with a 50 N load cell capacity in water at 37 °C. The upper clamp held the collagen membrane while the lower clamp held the nanofibrous scaffold. The force required to separate the layers and the strain at failure were measured at a speed of 10 mm/min and reported as mean ± SD.i is the initial dry mass of the samples before immersion in water and Mw is the mass of the hydrated samples.The in vitro swelling ratio of BSA-loaded scaffolds (5.2 × 5.2 cm) was measured using the mass change of samples after immersion in PBS for 5 min (n = 3). The swelling ratio was calculated from Equation (2):i is the initial dry mass of the samples before immersion in water and Mx is the dry mass after x time in PBS.BSA-loaded scaffolds (5.2 × 5.2 cm) were immersed in 8 mL of PBS at 37 °C under constant stirring. The samples were removed from PBS at different times up to 40 days, and sample remaining mass were measured following Equation (3):Two tests were performed, including the cytotoxicity of patch extracts as well as tests with cells in direct contact with the patches.4 cells per well (96-well plate) and allowed to attach for 24 h at 37 °C and 5% CO2. Then, the medium in contact with the materials was extracted and 100 µL was placed in contact with cells for an additional 24 h. After that time, the number of viable cells was obtained by a CellTiter Glo assay, based on quantification of the present ATP, which represents metabolically active cells.The in vitro cytocompatibility of patches was tested following EN ISO 10993-12 standards protocol (n = 3). BSA-loaded patches were decontaminated with UV-C irradiation . Scaffolds and control materials  were immerged in x ml of DMEM and were placed at 37 °C under agitation over 72 h. Meanwhile, L929 cells were seeded at 102 were UV decontaminated and fixed in 24-well plates (TCPS non-treated for cells culture) using silicone sinker. After 24 h of FBS starvation, 2 × 104 C2C12 cells at P6 were seeded on different samples and incubated in 1% FBS medium at 37 °C and 5% CO2. The number of cells was assessed after 1, 7, and 11 days of contact with the scaffolds using a PrestoBue assay that evaluates the transformation of weakly-fluorescent blue resazurin into highly fluorescent red resorufin through the mitochondrial activity of the cell.C2C12 myoblast proliferation on the nanofibers-collagen association samples was evaluated over 11 days. Scaffolds (n = 4) of 1.9 cm1H-NMR 2 as a catalyst. 1H-NMR  and SEC 1H NMR from the ratio between the methyne proton of the initiator and the methyne proton of the lactic unit were close to the targeted values (100 kg·mol−1). The compositions for the PLGA copolymers were also in good agreement with the initial monomers feed. SEC analyses showed a monomodal population with dispersity between 1.4 and 2.0. The molecular weights measured by SEC analysis were lower than those calculated by 1H NMR because of the amphiphilic nature of the Pluronic copolymers, which lead to changes of the hydrodynamic volume and therefore of the retention time.The copolymers molecular weights determined by BSA is classically used to protect growth factors during the electrospinning process . For thiThe SEM-EDX analysis b showed The release of BSA from the nanofibers composed of the different polymers was carried out to evaluate the release efficiency and to select the best polymers for further BSA/GF formulations. As shown in In more details, PLA nanofibers led to the lowest BSA release, with only 15% in 50 days. The introduction of the hydrophilic Pluronic block in the polymer increased the release efficiency with PLA-Pluronic-PLA nanofibers releasing 22% over the same period. Finally, the introduction of glycolic acid units (GA) further enhanced the release, as demonstrated by the cumulative release values of 25% and 36% obtained for PLAGA-Pluronic-PLAGA nanofibers containing 50 and 25% of glycolic acid units, respectively.w/w and PEG  at 14% w/w in the electrospun solution. Other porogen concentrations or even other porogens  were tested without positive results. Therefore, only the optimized formulations will be discussed.Considering the limited amount of BSA released after 50 days, we formulated nanofibers with additional porogen agents. Hydrophilic and biocompatible polymers with a similar molecular weight were chosen: Pluronic F127  at 29% 50 and PCL nanofibers, respectively.Following this first study of BSA release, two formulations have been selected for the inclusion of the growth factors IGF and HGF, namely PCL + Pluronic and PLA-Pluronic-PLA + PEG.As an important factor that influences protein diffusion from nanofibers, the swelling ratios of the nanofibers were evaluated e. The maHGF and IGF were encapsulated in PCL + Pluronic and PLA-Pluronic-PLA + PEG nanofibers. The surface morphology of the nanofibrous scaffolds was examined using SEM. As shown in HGF release b from PLWith the aim of this study being to develop collagen-based patches with GF-eluting properties suitable for cardiac regeneration, we have evaluated two methods of association of the loaded nanofibers with the collagen membrane. In the first method, named “sandwich association”, the nanofiber mat was embedded in a collagen sponge that adhered to the collagen membrane a. In theThe impact of the collagen association mode on HGF release was studied e. We obsWe wanted to verify that nanofibers were biocompatible. In view of the above results, we chose to focus on PLA-Pluronic-PLA + PEG patches that allow rapid release of IGF and sustained release of HGF in higher quantities than PCL + Pluronic patches. The percentage of viability of L929 in contact with PLA-Pluronic-PLA + PEG and PCL + Pluronic BSA-loaded nanofibers was 90% and 87% of the viability obtained on high density polyethylene film (negative control), which is higher than the 70% recommended by the European standard ISO 109993 a. This r50 , which is a resorbable polyester classically used for drug delivery and that is FDA approved [d,l-lactide from a Pluronic (F127) central block used as macro-initiator to yield PLA-Pluronic-PLA copolymers. In another approach, we also introduced glycolic units into the PLA blocks at different ratios to yield different types of PLGA-Pluronic-PLGA copolymers. Finally, we also used a commercial PCL whose high crystallinity and high hydrophobicity results in a much slower degradation rate than PLA [The objective of this work was to produce collagen-based patches with GF-eluting properties for at least 3 weeks that are suitable for cardiac regeneration. For this, we chose to encapsulate the growth factors in polymer nanofibers. Polymers were selected based on the criteria of compatibility with the electrospinning process, ease of processability, biocompatibility, degradability, and acceptability for biomedical applications. We focused specifically on the amorphous PLAapproved . In ordeapproved . We firsthan PLA . The rinAs growth factors are very sensitive, BSA is commonly used as a carrier to protect them during the electrospinning process . The proDue to their close molecular weight, few differences in BSA release kinetics were visible between the two porogens. However, the addition of porogens significantly reduced the amount of BSA still retained in the nanofibers after 35 days of release to values between 51 and 78%, compared to 64 and 85% without porogens, and thus increasing the release efficiency d. CorresFinally, two polymers were selected for further work: PCL + Pluronic and PLA-Pluronic-PLA + PEG, which reached, respectively, 50 and 34% of BSA release after 42 days. We studied the release of growth factors from these two types of nanofibers. The release rates of HGF were similar for both polymers, with a burst phase followed by a sustained release that reached 50% for PCL + Pluronic and 58% for PLA-pluronic-PLA + PEG b. These Although synthetic polymeric nanofiber scaffolds present great advantages owing to their good mechanical properties, excellent structural strength, and biodegradability, polymer nanofibers are not very favorable to cell adhesion. This is why polymer nanofibers have been associated in this work with a biomimetic material: collagen. Collagen alone presents relatively poor mechanical properties and is difficult to electrospin due to significant denaturation and degradation during electrospinning . ChemicaWe first associated a hydrated collagen membrane with HGF- and IGF-loaded nanofibers by simply placing the two materials in contact with each other and drying b. This sPrevious works have shown that IGF, as well as HGF, administered in bolus are bioactive from 10 ng/mL . Other s50-Pluronic-PLA50 + PEG. To improve the biocompatibility of the scaffolds, we then tested two modes of association of the growth factor-loaded nanofibers with a collagen membrane. We demonstrated that a hybrid patch associated with a collagen membrane through a collagen sponge allowed the sequential release of GF by delaying and then retaining the release of HGF over 35 days, while the release of IGF was rapid. The combination of collagen and growth factors improved the bioactivity on C2C12, making such multi-GFs releasing patches promising for cardiac regeneration.The objective of this study was to design cardiac regeneration patches that release growth factors for 3 weeks. In order to prolong the release of GFs, we encapsulated them in PLA- or PCL-based nanofibers. To this aim, various PLA copolymers were synthesized and evaluated with respect to the ability of the nanofibers to control GF release. Beside composition, the addition of macromolecular porogens in nanofibers was also investigated to improve the efficiency of release. This study led to the selection of nanofibers composed of PCL + Pluronic and PLA


The rise in antimicrobial resistance (AMR) is a major public health concern . A behavInaccurate penicillin allergy labels (PALs) are a huge burden on public health and have an adverse impact on AMS ,2. ApproThe aim of a direct oral penicillin challenge (DPC) is to determine if the patient is truly allergic to penicillin. Recent studies have highlighted the safety and feasibility of this intervention in a controlled secondary care environment following a relatively simple process of risk stratification involving a standardised drug allergy history . Penicilet al. [From an allergists' perspective, de-labelling with a prolonged course of an antibiotic is likely to enhance the sensitivity and the NPV of a DPC. The optimum duration of the antibiotic course for a DPC protocol, however, has not been established. Fransson et al.  highlighet al. .In patients with a history of a delayed onset reaction occurring on days 1–3 of a treatment course of penicillin, a 3 day DPC course may be sufficient, but in those reporting symptoms on day 5 or after, a 3 day course may be considered inadequate. Furthermore, some patients may not be fully reassured with a prolonged ‘sub-therapeutic dose’ owing to the fear about a reaction occurring at a higher dose.The majority of studies involving DPCs have used a ‘one size fits all’ approach with respect to employing the same dosing regimen regardless of the time of onset of symptoms of the index episode, making interpretation of published data somewhat challenging . A relatversus 3 days. In the same context, whilst single dose DPC is expected to have a lower risk of AMR, this needs to be balanced with potentially lower confidence in the NPV of the test. Patients with a PAL usually have a history of receiving alternative antibiotics [A further gap in the literature is the lack of evidence regarding the potential adverse impact on AMR in the context of a relatively prolonged DPC during opportunistic de-labelling. The risk of AMR is theoretically greater in patients undergoing opportunistic de-labelling; this may be further exaggerated by the duration of the DPC protocol e.g. 5 days ibiotics  and conshttps://www.who.int/news/item/01-10-2019-who-releases-the-2019-aware-classification-antibiotics). On balance, this may justify the acceptability of employing a prolonged DPC protocol with the ‘access’ group of antibiotics for de-labelling as opposed to a prescription of a ‘watch’ category antibiotic such as a carbapenem.The World Health Organisation (WHO) AWaRe classification categorises antibiotics as ‘access’, ‘watch’ and ‘reserve’. Amoxicillin, flucloxacillin or phenoxymenthylpenicillin are listed under the ‘access’ category of the WHO AWaRe classification. Antibiotics in the ‘reserve’ and ‘watch’ category are more likely to develop AMR (see Further research is needed to optimise and standardise DPC protocols. This should include qualitative studies involving healthcare professionals and patients to gain insight into their perspectives and behaviours. An ideal protocol will involve a dosing regimen that maintains high sensitivity and NPV for DPC in excluding an inaccurate PAL, whilst minimising the risk of AMR. In terms of opportunistic de-labelling, low-risk patients with a suspected history of an immediate reaction should be offered a single dose DPC ,3. HowevN/A.


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Antimicrobial resistance (AMR) is a challenge to human wellbeing the world over and is one of the more serious public health concerns. AMR has the potential to emerge as a serious healthcare threat if left unchecked, and could put into motion another pandemic. This establishes the need for the establishment of global health solutions around AMR, taking into account microdata from different parts of the world. The positive influences in this regard could be establishing conducive social norms, charting individual and group behavior practices that favor global human health, and lastly, increasing collective awareness around the need for such action. Apart from being an emerging threat in the clinical space, AMR also increases treatment complexity, posing a real challenge to the existing guidelines around the management of antibiotic resistance. The attribute of resistance development has been linked to many genetic elements, some of which have complex transmission pathways between microbes. Beyond this, new mechanisms underlying the development of AMR are being discovered, making this field an important aspect of medical microbiology. Apart from the genetic aspects of AMR, other practices, including misdiagnosis, exposure to broad-spectrum antibiotics, and lack of rapid diagnosis, add to the creation of resistance. However, upgrades and innovations in DNA sequencing technologies with bioinformatics have revolutionized the diagnostic industry, aiding the real-time detection of causes of AMR and its elements, which are important to delineating control and prevention approaches to fight the threat. In the year 1928, Alexander Fleming discovered penicillin, which killed the staphylococcal bacteria he was examining. It soon became the key antibiotic for the treatment of key diseases caused by Gram-positive pathogens . Apart fMicrobial pathogens continue to evolve and develop resistance to emerging and traditional treatment methodologies; and this, combined with the extreme decline in antibiotic research has only increased the magnitude of AMR and its impacts on global healthcare costs and outcomes. The rampant upswing in use of antibiotics with over-the-counter options has only fueled AMR. This also adds to the risk of re-emergence of many diseases; the emergence of XDR-TB, extensively drug-resistant tuberculosis, is an example worth citing.Antibiotics are a pillar of modern medicine. Their use has reduced childhood mortality. Beyond that, they are crucial for invasive surgery and complex treatments such as chemotherapy. They are irreplaceable aids in the complex era of robotics-guided surgeries, and for the management of secondary infections during sickness with the common flu, and infections of the skin, gut, etc. However, infections by multidrug-resistant bacteria are on the rise globally, causing the specter of untreatable infections to become a reality. A 2017 World Health Organization  report confirmed that the entire world would will run out of antibiotics, as the existing drugs in clinical use were developed through modifications to the existing classes, and have shown to have short impact cycles . The Eur(i)developing an enhanced derivative from an existing antibiotic family;(ii)designing structurally superior anti-bacterial agents ;(iii)identifying the benefits of alternative classes of agents, including phages.Monitoring the use of available antibiotics has been popularly recommended to tackle rising incidences of antibiotic resistance, apart from the pressing need for identifying new target molecules and redesigning of the existent one to tackle the mounting adversity from MDR and XDR pathogens. Different aspects that can be included in development include:One of the key strands of red-tape is regulatory aspects around certification, which adds to the complexity in the pipeline of developing new antibiotics, making it less lucrative for pharmaceutical companies to invest time and effort. The current situation indicates that urgently adopting new ways to fight antibacterial resistance is necessary. One such widely discussed approach includes the principle of nanotechnology, and the use of “nano functionalization” to re-establish the antimicrobial activity of existing antibiotics. A report by Natan, M. and Banin, E. 2017) details the use of nanotechnology to combat MDR, wherein nanoparticles (NPs) in two functional categories were introduced  details . These fTracanna, V. et al. (2017), in a report discussing the diversity of functions of prokaryotic antimicrobial compounds, highlighted different mining strategies, including ribosomal synthesis and post translational modification of peptides, polyketides, etc . These sThroughout history, the development of antibiotic resistance has been documented to be a natural process to enable survival of the bacteria, but has been fueled by human activities, including over-prescription and inappropriate prescription, overuse, excessive use of antibiotics as growth supplements in livestock, and the availability of few new antibiotics . NumerouAntibiotic resistance occurs when the target microbe develops a physiological mechanism to overcome the drug’s impact. This may be due to a change in bacterial envelope structure or composition, production of enzymes that break down the target agent, etc. Other mechanisms include limiting drug influx and increasing efflux, or modification/inactivation of drug target . Apart fAMR, apart from adding to the challenge around disease management, also impacts the patient. It has been documented to compromise the human immune system, and increase complications and vulnerability after complicated surgeries involving cancer, knee replacement, dialysis, etc. Further, individuals with comorbid conditions have an increased risk of severe adverse outcomes with AMR. Conditions which necessitate the use of “last-resort” antibiotics also significantly increase treatment costs to the client, prolonging in-hospital time and admission rates ,17.The mounting evidence around antibiotic usage practice being a crucial risk towards AMR necessitates the need for inculcating habitual and appropriately guided clinical management practices. Knowledge around vaccinations, transmission, and prevention strategies are the key in public health education. Elaborate care practices for wounds and infections among patients with comorbid conditions can reduce the burden on hospital admission, and control infection spread.Communicating the need for antibiotics based on diagnosis and recommended clinical management protocol is also a crucial aspect in AMR. A lack of diagnostic tools and regulatory guidelines, and self-treatment with over-the-counter antibiotics for ailments such as the common cold and flu are common in developing countries, adding severely to the burden of AMR . FurtherApart from changes in antibacterial use and consumption patterns across different global economies, modern day travelling has also been a major contributor towards dissemination of new infections and antibiotic resistance across the world. The recent coronavirus disease (COVID-19) pandemic is the best example. One documented study among European travelers from India identified the presence of carbapenemase-producing Enterobacteriaceae (CPE), even among those with no contact with the Indian healthcare system during their stay ,21.Transmission of highly drug resistant microbes has been documented to occur inter- and intra-species. Identifying the reservoirs and best practices around causes for transmission will be fundamental to controlling future pandemics. Pandemics and epidemics involving COVID-19 are examples involving viruses which spread material between species. Any public healthcare measure that can control the dissemination and use of antibacterial agents is the first step in controlling AMR. High-quality global surveillance systems are needed to provide warnings associated with changes in antimicrobial use, and avoidance of the time lag in knowledge transmission is a key to preventing a global health crisis by AMR.Preventing infections and controlling transmission.Improving antibiotic use to slow the development of resistance through high quality surveillance and usage guidelines.Stopping the spread of resistant microbes when they do develop through antimicrobial stewardship programs.Antibiotic resistance development is a gradual process wherein the microbes in question develop resilience against agents targeted towards their destruction. Addressing this threat needs aggressive action towards:The science of the gut microbiome has presented ever-expanding opportunities to study the human—microbiome interaction, and provide an ecosystem to assess microbiome—antibiotic interactions. The impact of antibiotics on the human gut’s microbiome continues to be a key topic. Antibiotic exposure in early childhood has been linked to immunological, neurological, and gastrointestinal impacts; and antibiotic use at any age has been linked to negative impacts on the gut microflora leading to the emergence of antibiotic-resistant diarrhea . Apart fAntibiotic-induced microbiome depletion is a well-documented phenomenon due to antibiotic therapy and has been implicated in the alteration of metabolic homeostasis through gut signaling . StudiesClostridioides difficile infection (CDI), with which cure rates of up to 90% have been recorded. Other conditions include inflammatory bowel disease, with the respective clinical efficacy ranging between 25 and 50%. The differences in the impact of FMT have also been attributed to differences in microbiome composition between donors and recipients, due to changes in diet and environment at different time points . Though the panel is often predefined for a set of ARGs, the high confidence prediction makes it suitable for clinical use.The fundamental approach to identifying the presence of ARGs involves studying the whole genome sequencing data, for both the endogenous elements and plasmids. The data on presence/absence and abundance of vectors have been used for the development of machine learning models to predict the occurrence of AMR in samples. A report on prediction of AMR by WGS in lgorithm . Anotherlgorithm . PhyResSiagnosis . The tooiagnosis . The “MyE. coli. This study found acquisition of resistance to one drug changes susceptibility and resistance to others. Subsequent gene expression analysis led to the identification of dynamic compensatory mechanisms. Teixobactin, a new antimicrobial agent has documented activity against S. aureus and Enterococcus faecalis without resistance. A study which reported on the transcriptional response of E. faecalis to teixobactin levels identified development of intrinsic tolerance to high concentrations of the same (through deletion of croRS), and this is generally considered a precursor for the development of resistance [E. coli, and identification of differentially expressed transcripts, are further delayed by the need for functional annotation to decipher their role in AMR.Another approach to AMR prediction is through differences in gene expression of the isolate post exposure to the target drug. A report by Suzuki S et al. , detailssistance . BesidesM. tuberculosis and S. aureus as a powerful approach. This study highlighted the significance of genotyping with k-mers, and the benefits include being alignment-free and the ability to identify different categories of genetic variants, including single nucleotide insertions and deletions in the coding and non-coding regions. The study identified k-mers , which were further used in linear regression models for each tested antibody and found to have performance on par with Mykrobe [This approach elucidates AMR mechanisms based on global genomic comparisons with various susceptibility profiles of multiple strains. Another proof-of-concept study recorded the significance of stability selection to investigating the bacterial genotype–phenotype relationship in  Mykrobe .M. Tuberculosis to identify genetic signatures of antibiotic resistance. Subsequent mapping to available 3D structures of select proteins associated with AMR allowed the authors to pinpoint mutation clusters of certain proteins in the antibiotic response. This insight may help to better explain the AMR causality of alleles [Studies have highlighted the significance of machine learning with pan-genome structural analysis of  alleles . Another alleles .The pan-genome approach is funding intensive due to need for genomic sequencing technology and tools for a genome-wide comparison approach, although it holds great promise for clinical applications. An advantage is its ability to explore entire genomes instead of known AMR-related regions, to identify new mechanisms or regulatory elements. This is crucial to determining if the relation between a genomic signature and AMR is a statistical probability or causal. Furthermore, the need for large collections of strains is key to obtaining informative insights into AMR, which adds to the challenge.E. coli and simulated the corresponding metabolic status via genome-scale metabolic network model. The white-box machine learning approach yielded information on pathway mechanisms, facilitating the quantification of the relative contributions of each metabolic pathway towards tackling lethal mechanisms of each antibiotic. Further, profiling of over 500 intracellular and extracellular putative metabolites involving the 190 evolved populations highlighted carbon and energy metabolism to constrain the speed and mode of resistance acquisition. The study further identified multiple mechanisms employed by the E. coli strain to compensate for the pressure from different antibiotics, including mutations or expression of efflux pumps, which was further found to be influenced by the carbon source, and therefore, the metabolic state the organism is in [Studying metabolic profiling has also been widely studied as an approach orthogonal to the ones discussed previously. A report by Yang JH et al.  discussesm is in . This apResistance towards antibiotics is an inevitable part of microbial evolution. This emphasizes the need for surveillance of ARGs as the key aspect in future policy making. WGS and metagenomics, coupled with powerful bioinformatics tools and databases, have only furthered our knowledge in this regard. The reliability of results from these techniques foster encouragement for their execution in routine diagnostics. Use of antibiotics leads to dysbiosis of the gut microbiome, causing several side effects. In the monitoring of AMR in general and in the implementation of NGS technologies in particular, each domain has its own set of technical challenges, requirements, and realities. The only way forward is continuous education, surveillance, data analysis, and research to tackle the ever-emerging AMR crisis. A global public awareness campaign tackling the supply problem, control of antibiotic usage, and stimulus to the diagnostic market to initiate more rapid diagnostic tests, has been discussed as a key intervention with which to tackle AMR.The bioinformatics approach to AMR identification includes the aspects that focus on reliable insights of AMR that are replicable in clinical settings. This is focused towards underlining new genes of resistance, the regulatory mechanisms, or new antibiotic targets. Every approach has limitations which should be taken into consideration when building and training superior prediction models. The combination of advanced molecular technologies and powerful artificial intelligence and machine learning algorithms holds great promise for understanding AMR at the molecular level to generate clinically relevant predictions, leading towards selection of personalized antibiotics geared for favorable clinical outcomes.


Due to a production error, there was an error in affiliation 1. Instead of “Key Laboratory of Molecular and Cellular Biology of Ministry of Education, Key Laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province, College of Life Sciences, Shijiazhuang, China”, it should be “Key Laboratory of Molecular and Cellular Biology of Ministry of Education, Key Laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province, College of Life Sciences, Hebei Normal University, Shijiazhuang, China”.The publisher apologizes for this mistake. The original version of this article has been updated.


Lupinus luteus L.) and its Alcalase hydrolysates were examined for their functional and antioxidant properties in relation to surface hydrophobicity of proteins and peptides and molecular weight distribution. Enzymatic hydrolysis improved the foaming characteristics of lupin proteins, while the emulsifying properties deteriorated. It means that good foaming properties of preparations are determined by the presence of low-molecular δ conglutin and small subunits of γ conglutins. In turn, larger proteins such as α and β conglutin are responsible for maintaining good emulsifying properties. The measured surface hydrophobicity was consistent with the results of emulsifying properties. It has also been shown that the scope of changes in antioxidant properties due to hydrolysis, measured by DPPH method and as reducing power, is more pronounced than with the use of ABTS and FRAP methods.Protein isolate obtained from sweet yellow lupin ( The use of legume seeds as a source of food protein is of growing interest to consumers, especially in view of the increasing trend towards vegetarian diet. From a global perspective, soybeans are traditionally the most popular protein raw material from this group of plants, although consumers also appreciate legumes such as chickpea, pea, and lentils.Relatively less attention is paid to lupin, which is after all a valuable legume with high protein content, comparable with that in soy. The protein quality of its seeds is equally good and rated by some authors as better than soybeans. Although it is poor in sulfur-containing amino acids, it also has less antinutritional factors than soybeans—trypsin inhibitors and hemagglutinins are practically absent –3.Lupin is also easy to grow because it tolerates frost, drought, and poor soils quite well. However, although known as human food since early Roman times, it is utilized to a small extent so far, primarily because of alkaloid content and low agronomic yield. Commonly found in lupins are quinolizidine alkaloids such as lupanine, sparteine, or lupinine, which have been shown to be reversible inhibitors of acetylcholinesterase. Alkaloid level depends on cultivar, soil type, and growing season. Some cultivars have been developed, however, which are sweet and contain alkaloids at the level safe for human health , 4, 5. MLupinus angustifolius protein. In turn, Surówka et al. ; 10 g/L) was mixed, and the whole was incubated at 50°C for 30 min. Subsequently, 2.5 mL of trichloroacetic acid solution (100 g/L) was added to the mixture and vortexed. Afterward, the entire mixture was centrifuged at 4500 rpm for 15 min. The supernatant (2.5 mL) was transferred to a tube, and 2.5 mL of distilled water was added along with 0.5 mL of iron (III) chloride solution (1 g/L). After 7 minutes, the absorbance at 700 nm was measured, relative to a blank in which the extract was replaced with distilled water. Results were converted to ascorbic acid equivalents (mg/100 mL), based on the standard curve.The volume of 1 mL of extract, 2.5 mL of phosphate buffer , and 2.5 mL of potassium ferrocyanide (KP < 0.05 using Duncan multiple range test. Data were presented as means ± standard deviation (SD).All experiments except for electrophoresis were carried out in triplicate. Results of measurements were statistically analysed by analysis of variance (ANOVA) using the CSS Statistica v. 12 package . Differences were considered significant at In vegetable protein products protein concentration, molecular weight distribution and surface hydrophobicity determine their functional properties.P < 0.05). Schlegel et al. [In this study, lupin protein isolate, with 87.9 ± 0.2% protein content, was produced from yellow lupin flour containing 39.3 ± 2.4% protein, by means of alkaline leaching and isoelectric precipitation. El Adawy et al.  receivedβ- and 11S-α-globulins are predominant in lupin flour proteins. This corresponds to the SDS-PAGE results, since the proportion of protein fractions of 42-66 kDa was the highest (30.7%) in the analysed nonhydrolyzed LPI. It is assumed that within this range, there are subunits of one of the two main globulin fractions: legumin-like α conglutins (11S-α-globulins with above 330 kDa molecular weight) and vicilin-like β conglutins (7S-β-globulins with molecular weight of 143-260 kDa). Legumins are hexamers composed of heterogeneous acidic subunits (42-52 kDa) and basic subunits (20-22 kDa), while vicilins are trimers consisting of three subunits, which molecular weights range between 17 and 64 kDa  [The quantitative results of SDS-PAGE separation of proteins in LPI and hydrolysates are shown in -20 kDa) . Since tδ conglutins (2S globulins) with molecular weight of about 13 kDa, composed of subunits: small (4 kDa) and large (9 kDa) [β conglutins [The next important protein fractions of the LPI (17%) were low-molecular-weight proteins below 15 kDa. These proteins are probably part of the  (9 kDa) . In thisnglutins , 28. A bAnalysis of the obtained electrophoretic data showed that some fractions were occurring only in individual lines, and there were others, which repeated in all samples–namely in LPI and hydrolysates. The repetitive fractions visible on the gel scan  were maiLupinus angustifolius L. in order to reduce the abundance of major allergens and concluded that Alcalase was effective in the hydrolysis of the high-molecular-weight fractions of α conglutin and β conglutin, as well as the medium molecular weight fractions of LPI.In hydrolysates, the main fractions (above 70%) were proteins with a molecular weight below 15 kDa. The relative content of these low-molecular-weight fractions in enzyme hydrolysates was 4-5 times higher than their level in the raw material (LPI). Similar results of the hydrolysis with Alcalase were obtained by Schlegel et al.  who, as β conglutin (53-66 kDa) and acidic α conglutin (42-52 kDa) subunits, as well as in other polypeptide chains of molecular weight above 15 kDa, was observed. This is due to the breakdown of proteins into low-molecular-weight peptides and amino acids. However, compared to other bands, the loss of the fractions 42-66 kDa and 20-22 kDa is relatively less than, for example, fractions with molecular weights in the 34-40 kDa, 26-34 kDa, or 18-20 kDa range. This suggests a greater susceptibility of IMW and LMW subunits of β conglutin and large subunits of γ-conglutin to the proteolytic activity of Alcalase in comparison to the other fractions of LPI. Present after hydrolysis with Alcalase but faintly marked bands of about 20-22 kDa, probably originated from 11S globulins, were also observed by Karamać et al. [In our study, when comparing the LPI and products of its modification with Alcalase, a significant decrease in the content of the high-molecular-weight (HMW) ć et al. , who stuć et al. , who stuAnalysis of protein band percentages showed differences between individual hydrolysates, which may affect differences in the examined functional properties. The product obtained as a result of two-hour proteolysis (H-120) had higher content of low-molecular-weight protein fractions (below 15 kDa) than H-30 and H-60 hydrolysates.In order to identify the products of proteolysis more accurately, the separation of peptides was performed according to the Schägger and von Jagow procedure , using aThe electrophoretic analysis by means of the Schägger and von Jagow procedure revealed that proteins with molecular weights in the range of 43-65 kDa (more than 26%) were predominant in LPI, and this result confirmed the findings obtained by the standard SDS-PAGE electrophoresis. With regard to hydrolysates, however, the predominant protein fraction changed with a prolongation of the proteolysis length. It was found that in hydrolysates H-30, H-60, and H-120, the dominant compounds were those with molecular weight of 43-65 kDa, 24-33 kDa, and below 10 kDa, respectively. The bands that represented the low-molecular-weight fractions in these preparations were wide and fuzzy. The observed phenomenon is a result of polydispersity, i.e., an approximately continuous molecular weight distribution, with no dominant bands corresponding to a higher content of the molecular weight fraction.P < 0.05) and assumed similar values to each other (5.90–6.69), showing no statistically significant differences (P > 0.05). This indicates that the initial hydrolysis of lupin proteins is already enough to reduce this parameter significantly; the prolongation of the process does not change it substantially.The results obtained for the surface hydrophobicity of lupin protein preparations, presented as a plot and the simple equations fitted to them, are shown in Similar results of reducing surface hydrophobicity were obtained by Surówka et al. , who hyd5 value increased, which means that foam stability decreased as compared to the LPI. Moreover, this decrease seems to be to some extent related to the length of the process. Therefore, in order to improve the foam stability of hydrolysate solutions, they should be used in preparations with stabilizing substances, e.g., polysaccharides increasing the viscosity.In foams characterized by high FO values, gas bubbles are large, and/or the foam walls are relatively thin. Thus, high FO values indicate a technologically unfavourable structure of the foam. As for hydrolysate preparations, the FO values decreased significantly compared to the FO values determined for the LPI. As a result, the foam obtained from the hydrolysate solutions had a desirable, more finely porous structure with strong walls. As in the case of FC, an improvement of this aeration property was noted due to enzymatic hydrolysis. However, according to the data in δ conglutins, the LMW subunits and small subunits of β and γ conglutins had better adsorption capacity on the surface of air bubbles, providing good foaming properties to these products. Peptides present in hydrolysates can reach the interfacial surface much faster than proteins and create films that form the foam structure. The above authors concluded that degree of hydrolysis determines the foaming capacity of hydrolysates; with an increase in the degree of hydrolysis and a decrease in the average molecular weight, the ability to form foams increases. This is due to the fact that proteins with lower molecular weight have a greater ability to adsorb on the surface of air bubbles. Polar moieties located on the surface of the protein molecule more easily turn towards liquid and nonpolar ones towards the air. As a result, a coherent, flexible film forms around the air bubbles.According to Surówka et al. , who invEmulsion with the use of LPI is formed relatively efficiently as measured by EAI . This inAlcalase hydrolysis had an even greater effect on ESI. While the emulsions obtained from LPI showed good stability, as a result of the hydrolysis, the ESI index decreased several times .It is believed that the surface hydrophobicity of proteins affects the emulsifying properties , 34. Wheα and β conglutins fragmentation, the number of peptides increased, but the number of both hydrophobic and hydrophilic moieties in their molecules was reduced. Even when the peptides are positioned on the surface of the dispersed lipids, interactions with the aqueous phase, which stabilize emulsions, will not be formed. In consequence, a loss is observed of emulsifying properties and decreased stabilization of the emulsion. Therefore, among the analysed preparations, LPI showed better emulsifying properties than hydrolysates. Similar observations were reported in studies on rape protein hydrolyzed with Alcalase, where a decrease in emulsifying capacity as a result of hydrolysis was observed [In this study, as a result of observed .The use of four methods to measure antioxidant activity allows for a more holistic determination of the total antioxidant capacity of a given compound or group of compounds, as there are some limitations for each analysis that may disturb the correct assessment of the antioxidant activity of a given sample if it were evaluated by only one analytical method , 37. AmoThe ABTS and DPPH methods are based on both SET (single electron transfer) and HAT (hydrogen atom transfer) mechanisms, whereas the determination of antioxidant activity by the FRAP and reducing power measurement assays is based solely on the HAT mechanism .Analyses of antioxidant activity performed by four various methods showed that the results obtained were strongly dependent on the measurement methods Figures  and 6. LChanges in the length of polypeptide chains caused by hydrolysis and the accompanying significant decrease in surface hydrophobicity affected substantially the examined functional properties. This was manifested by the reduced emulsification capacity of hydrolysates and much poorer emulsion stability compared to LPI. The peptide chains formed as a result of proteolysis were too short, and hydrophobic domains were exposed insufficiently to create the film on the interface stable enough to limit coalescence.On the other hand, it was observed that along with the prolongation of the hydrolysis time and the decrease in the protein average molecular weight, the foam capacity of hydrolysates was improved, although stability of the foam decreased.δ conglutin, LMW subunits of vicilin-like β conglutins, and small subunits of γ conglutins.In comparison with the hydrolysis products, LPI was characterized by much lower foam formation capabilities. The improvement of foaming properties due to two-hour proteolysis was connected with the presence of low-molecular When considering antioxidant properties, LPI was characterized by a distinctly better antioxidant activity than hydrolysates, as measured by the DPPH method and as reducing power, whereas the results obtained by the ABTS and FRAP methods were comparable.


Background: Understanding the temporal trends in the burden of lower respiratory tract infections (LRI) and their attributable risk factors in children under 5 years is important for effective prevention strategies. Methods: We used incidence, mortality, and attributable risk factors of LRI among children under 5 years from the Global Burden of Diseases database to analyze health patterns in 33 provincial administrative units in China from 2000 to 2019. Trends were examined using the annual average percentage change (AAPC) by the joinpoint regression method. Results: The rates of incidence and mortality for under-5 LRI in China were 18.1 and 4134.3 per 100,000 children in 2019, with an AAPC decrease of 4.1% and 11.0% from 2000, respectively. In recent years, the under-5 LRI incidence rate has decreased significantly in 11 provinces  and remained stable in the other 22 provinces. The case fatality ratio was associated with the Human Development Index and the Health Resource Density Index. The largest decline in risk factors of deaths was household air pollution from solid fuels. Conclusions: The burden of under-5 LRI in China and the provinces has declined significantly, with variation across provinces. Further efforts are needed to promote child health through the development of measures to control major risk factors. Lower respiratory infection (LRI), defined as pneumonia or bronchitis, is the leading cause of death worldwide, causing more than 2 million deaths in 2019 . The ratA handful of global initiatives, such as the Stop Pneumonia Initiative, the Integrated Management of Childhood Illness initiative, and the Global Action Plan for the Prevention and Control of Pneumonia and Diarrhoea (GAPPD), provided guidance on the most effective interventions to avoid illness and death caused by LRIs ,6,7,8.GABy the end of the 20th century, pneumonia was the first and second leading cause of death among children under 5 years in rural and urban areas of China, respectively . In the http://vizhub.healthdata.org/lbd/lri, accessed on 1 January 2022) and the National Bureau of Statistics . World Bank data showed the Human Development Index (HDI). We used mortality and incidence data from GBD 2019 to estimate trends of the burden for LRI among children under 5 years old in China. The NBS population data were used to estimate the amount of incidence and deaths from LRIs in 2019. These data were from 33 provincial administrative units in China, including 22 provinces, 4 municipalities, 5 autonomous regions, and 2 special administrative regions. Estimation of the rates of mortality and incidence for under-5 LRI included four age groups: 0–6 days , 7–27 days , 28–364 days , and 1–4 years. LRIs were defined as diseases of the lower respiratory tract, including pneumonia and capillary bronchitis, which were subdivided into A48-1, A70, B96-0-B96-1, B97-21, B97-4-B97-6, J09-J18-2, J18-8-J18-9, J19-6-J22-9, J85-1, J91-0, P23-P23-9, U04-U04-9, and Z25-1 in the International Classification of Diseases 10th revision (ICD-10) [The main sources of data were the Global Burden of Disease  .The detailed methodology for GBD 2019 and LRI incidence and mortality estimation in GBD 2019 was published previously ,14. DataThe Health Resource Density Index (HRDI) was calculated as the geometric mean of health resources per 1000 people per square kilometer . The folYi represents the health personnel resource of unit i; Pi represents the population of unit i; and Ai represents the area of unit i.The case fatality ratio (CFR) is defined as the ratio of the number of deaths to the number of incident cases. The associations between HDI, HRDI, and CFR of LRI for each province were examined by linear regression analysis. The corresponding 95% confidence intervals (CIs) were generated from the regression.Joinpoint regression enables one to test whether the trends are statistically significant throughout the observation period and provides a straightforward and interpretable output in terms of annual rates of change . JoinpoiThe risk factors for LRI mortality rate were modelled independently as part of the comparative risk assessment framework in the GBD study ,19. The p-values less than 0.05 were statistically significant.All pictures were drawn using Python 3.8 (an open source software) and ArcGIS 10.8 . Gender differences were tested using chi-square tests. All The Chinese LRI incidence rate was 4134.3  and Xizang . The mortality rate declined significantly from 2000 to 2019 , Yunnan , and Sichuan , second-hand smoke , and ambient particulate matter pollution (3.5 deaths, 95% UI: 2.4~5.0, per 100,000 children; We performed a thorough and up-to-date evaluation of the burden and epidemiologic temporal trends of LRI for children under 5 years at province level in China. The burden of LRI among children younger than 5 years declined substantially in all provinces from 2000 to 2019, while the declines did not occur equally across provinces. HDI and HRDI were strongly associated with the CFR of LRI. Notably, it is possible to well manage LRIs for children under 5 years of age by effectively controlling the risk factors.We found that the LRI mortality rate declined more rapidly than incidence rate in Chinese provinces, indicating that improvements in protecting against death may have outweighed improvements in reducing the risk of potential infection, which is in concordance with previous global evaluation . China hThere was some variation in provincial trends in the incidence rate of under-5 LRI. Zhejiang, Shanxi, Ningxia, Hubei, and Hong Kong showed a meaningful increasing trend in the most recent years of LRI incidence rate among children aged 1–4 years, while the other provinces showed a decreasing or stable trend. Policies to balance inequalities across regions have been a priority for the Chinese government , and ourIdentifying risk factors is important for the prevention of LRI. In 2019, the major risk factors for LRI-related deaths in China included child wasting, ambient particulate matter pollution exposure, and second-hand smoke. Children’s respiratory systems are more vulnerable to the adverse health effects of air pollution, due to the immaturity of the lungs and immune system . ChildreWe sought to provide the latest estimation of under-5 LRI burden for different regions in China in order to advance evidence-based prevention programs. Although previous studies have analyzed trends in under-5 LRI in China, we filled a gap in the study by focusing on provincial time trends in LRI among children under 5 years of age ,12. On tOver the past two decades, the burden of under-5 LRIs in China has been declining. However, the rate of decline in the burden of LRIs has slowed in recent years. The Chinese health system should focus on new approaches to LRI prevention, including: providing greater access to healthcare, expanding interventions, addressing in-country disparities, and further efforts to focus on the management of LRI risk factors to reduce preventable deaths from LRI in children under 5 years of age. In addition, further epidemiological studies, including more detailed studies of subgroups and risk factors by province, are needed for future research.


The antimicrobial resistance (AMR) crisis from bacterial pathogens is frequently emerging and rapidly disseminated during the sustained antimicrobial exposure in human-dominated communities, posing a compelling threat as one of the biggest challenges in humans. The frequent incidences of some common but untreatable infections unfold the public health catastrophe that antimicrobial-resistant pathogens have outpaced the available countermeasures, now explicitly amplified during the COVID-19 pandemic. Nowadays, biotechnology and machine learning advancements help create more fundamental knowledge of distinct spatiotemporal dynamics in AMR bacterial adaptation and evolutionary processes. Integrated with reliable diagnostic tools and powerful analytic approaches, a collaborative and systematic surveillance platform with high accuracy and predictability should be established and implemented, which is not just for an effective controlling strategy on AMR but also for protecting the longevity of valuable antimicrobials currently and in the future. The increasing prevalence of antimicrobial resistance (AMR) among bacterial pathogens poses one of the greatest threats to public health all over the world , mainly To identify common AMR and guide diagnostics and effective treatment for bacterial infections, a gold-standard laboratory test used to determine the antimicrobial sensitivity of AMR organisms, termed antimicrobial susceptibility tests (AST), has been adopted in clinics for over 50 years. Traditional AST involves broth microdilution and disc diffusion on agar to determine the minimum inhibitory concentration (MIC). The MIC value indicates the lowest concentration of antibiotics where bacterial growth stops, and the growth level is usually measured by absorbance or light scattering. Before and during testing, multiple turnarounds of culturing the bacteria in the sample and isolating colonies and growth needed to be accomplished before AST. The whole pipeline takes at least 48 h, which usually provides the results after using an empirical prescription of antibiotics that might be misused. It has been estimated that around one-fifth of prescribed antibiotics from 2013 to 2015 were inappropriate in the United Kingdom . Also, cPseudomonas aeruginosa exhibit high plasticity of resistance phenotype driven by environmental changes  for AMR diagnosing has been considered an efficient avenue for combating AMR . The gen changes . Overall changes . AMR bacThe COVID-19 pandemic has seriously affected global efforts against AMR , magnifyYersinia pestis  by determining the cellular fluorescence intensity owing to antibiotic-caused membrane damage and the resulting uptake of propidium iodide or rose Bengal , aacA-aphD (aminoglycoside resistance), tetK, tetM (tetracycline resistance), erm(A), erm(C) (macrolide-lincosamide-streptogramin B resistance), vat(A), vat(B), and vat(C) (streptogramin A resistance) , AMR genes , and even could detect cancer mutations with attomolar sensitivity and single-base mismatch specificity  or RT-PCR , microflistance) . With thn 25 min ; 10 copiated SNP ; the majar assay . The twocificity . This CRo copies  and idenrm genes . Curti ehttps://joinarmada.org/), a nonprofit global organization combatting superbugs with a database of bacterial genomes, has been established to create a specific “criminal database” of AMR pathogens, collecting an unprecedented amount of bacterial strains and details of their antibiotic resistance profiles, genetic identity and epidemiology from a global network of hospitals, veterinarians, scientists, and other advocates. WGS emerges as a powerful tool for understanding the genetic makeup of bacteria AMR. As the high-throughput sequencing technology developed in the mid-2000 s, it can illustrate a landscape of the whole resistome in a couple of days with a sophisticated downstream bioinformatic pipeline . The newLearning essential characteristics directly from the data of genomic sequences, machine learning obviates the need for prior knowledge of resistant strains caused by unknown mechanisms. It has been proven valuable for predicting AMR with an unbiased method . Unlike S. aureus  were selected to determine the susceptibility by performing quantitative RT-PCR, and the results are consistent with MIC values. in situ hybridization and immunofluorescence tests to locate and quantify the mRNA and protein existence of the TEM β-lactamase, conferring ampicillin resistance in the E. coli. Campylobacter jejuni and analyzed the data in the Comprehensive Antibiotic Resistance Database for AMR detection. It was found that both genomic and proteomic approaches can identify molecular determinants responsible for resistance to tetracycline and ciprofloxacin, in line with their phenotypes. These methods require several steps and analyses, which are more suitable for basic research, but not feasible for the field application. For rapid RNA detection, the RNA-targeting CRISPR-associated enzyme Cas13a directly binds to the target RNA and releases the positive signal (The DNA-based method only detects the existence of these potential resistance DNA traits without providing information regarding efficient transcription, expression, and antimicrobial susceptibility. The feedback in bacterial transcriptome and proteome to antibiotics exposure offers real-time dynamic information of relevant genes. Their expression change in mRNA and protein levels is endowed with key diagnostic values for AMR detection. Some efforts have also been made in transcriptome and proteome, but remain limited. e signal . Combinee signal . In clinA comprehensive analysis has suggested more factors to make more precision diagnostics and informed preclinical decisions . To dealFrom the clinical perspective, the priority is confirming whether antimicrobial therapies are needed in a particular case; the second is which drug is suitable for an optimized treatment if a bacterial infection is already present. Apparently, the most clinical urgency for patients and physicians is not knowing which bacteria is resistant to what antimicrobial but determining which narrow-spectrum drug can eliminate the pathogen. Most diagnostic methods aim to identify drug-resistant determinants of superbugs, but few studies explored if there are any drug-susceptible determinants or other multi-omics traits in AMR strains. These drug-susceptible feature data can be used for model training in machine learning, and molecular-based assays can detect drug-susceptible markers directly. More innovative efforts are needed in this area in the future.In the coming decade, advancements in technical and computational tools for multi-omics approaches are continually improving knowledge regarding the diverse AMR mechanisms and integrating a deep mechanistic understanding of AMR determinants with a broader systematic analysis of microorganisms. The ultimate goal will lead to a revolutionary change in AMR diagnostics, significantly guiding the surveillance of AMR threats and finally slowing down the rising crisis.


Circadian photoperiod, or day length, changes with the seasons and influences behavior to allow animals to adapt to their environment. Photoperiod is also associated with seasonal rhythms of affective state, as evidenced by seasonality of several neuropsychiatric disorders. Interestingly, seasonality tends to be more prevalent in women for affective disorders such as major depressive disorder and bipolar disorder (BD). However, the underlying neurobiological processes contributing to sex-linked seasonality of affective behaviors are largely unknown. Mesolimbic dopamine input to the nucleus accumbens (NAc) contributes to the regulation of affective state and behaviors. Additionally, sex differences in the mesolimbic dopamine pathway are well established. Therefore, we hypothesize that photoperiod may drive differential modulation of NAc dopamine in males and females. Here, we used fast-scan cyclic voltammetry (FSCV) to explore whether photoperiod can modulate subsecond dopamine signaling dynamics in the NAc core of male and female mice raised in seasonally relevant photoperiods. We found that photoperiod modulates dopamine signaling in the NAc core, and that this effect is sex-specific to females. Both release and uptake of dopamine were enhanced in the NAc core of female mice raised in long, summer-like photoperiods, whereas we did not find photoperiodic effects on NAc core dopamine in males. These findings uncover a potential neural circuit basis for sex-linked seasonality in affective behaviors. Day length, or photoperiod, is a reliable indicator of the changing seasons, and is a powerful environmental cue influencing affective state and behavior. While studies have shown that photoperiod can modulate neurotransmitter systems involved in affective behavior, like the dopamine system, there are few studies examining effects of photoperiod on the synaptic level. Here we assess mesolimbic dopamine dynamics in mice maintained in seasonal photoperiods. We found a sex-specific effect wherein photoperiod modulates dopamine release from and uptake in dopaminergic terminals in the nucleus accumbens (NAc) core of female mice with long summer-like photoperiods increasing dopamine release, thus uncovering a potential synaptic basis for sex-linked seasonality in affective behaviors.Circadian photoperiod, or day length, is an environmental signal that synchronizes daily biological rhythms to local time and induces seasonal changes in behavior and physiology . While sDopamine is a neuromodulator that plays a critical role in regulating affective state and state-dependent behaviors . More spDopamine signaling in the NAc is tightly and dynamically regulated through both VTA neuron activity and intra-accumbal mechanisms at dopamine axon terminals . VTA dopSex differences in the function of the mesolimbic dopamine pathway are well established , making Here, we assess the effect of seasonal photoperiods on NAc dopamine dynamics in mice using fast-scan cyclic voltammetry (FSCV). We found a sex-specific effect wherein photoperiod modulates dopamine release and uptake in the NAc of female mice. Specifically, female mice raised in long summer-like photoperiods exhibit greater dopamine release, thus uncovering a potential synaptic basis that provides insight to sex-linked seasonality of affective behaviors.+/+ mice for all experiments. C3Hf+/+ mice endogenously synthesize melatonin and lack the retinal degeneration alleles of the parent C3H strain and are therefore responsive to photoperiod manipulation . Mice were humanely killed and the brain was rapidly removed. Using a Leica Vibratome, 250-μm-thick sagittal sections containing the NAc core were collected from whole brain tissue in oxygenated  artificial CSF (aCSF) containing (in mm): 126 NaCl, 2.5 KCl, 1.2 NaH2PO4, 2.4 CaCl2, 1.2 MgCl2, 25 NaHCO3, 11 glucose, and 0.4 L-ascorbic acid, and pH adjusted to 7.4. Slices were transferred to a chamber containing oxygenated aCSF. All experiments were performed using a Scientifica SliceScope Pro System in 32°C aCSF with a flow rate of 2 ml/min. The carbon fiber microelectrode  and bipolar stimulating electrode were placed in close proximity in the NAc core. A single electrical pulse  was applied to the tissue every 4 min to evoke dopamine release. We also evoked dopamine release using a train of five pulses at 5, 10, or 20 Hz . Extracellular dopamine was recorded by applying a triangular waveform .m, 1 μm, and 3 μm concentrations. We collected peak evoked dopamine release at each concentration until stable responding was achieved (three collections with <10% variability) before moving onto the next highest concentration.For pharmacological experiments using DAT inhibitor, GBR 12783 (Tocris Bioscience/Bio-Techne), peak evoked dopamine release was collected in aCSF until a stable baseline was established (three collections with <10% variability) using a single electrical pulse every 4 min. GBR 12783 was bath applied to the slice at 300 nVmax). As described previously  to a known concentration of dopamine (3 μm) via flow-injection system, and we used these values to convert current to dopamine concentration.Demon voltammetry and analysis software was used to analyze all FSCV data . Data coeviously , Km from methods , and bas methods , we set p values < α indicating a statistically significant difference. Statistical significance was determined by two-way ANOVA. All post hoc analysis was performed using Tukey’s multiple comparison tests and standard error of the mean was used for all experiments. Power analyses were performed with preliminary data during the acquisition of each new data set. The sample size obtained from each power analysis calculation was then compared with sample sizes reported in the literature for similar experiments. Errors bars depicted in figures represent SEM.Prism 9 (GraphPad Software Inc.) was used for all statistical analyses. For all analyses, α was set as 0.05, with ex vivo slices from both male and female mice raised in Short (8L:16D), Equinox (12L:12D), or Long (16L:8D) photoperiods (N = 9), Equinox (N = 12), and Long (N = 10) where N is number of animals. To assess whether photoperiod differentially affected dopamine release from tonic and phasic firing modes of dopamine neurons projecting to the NAc core, we used a range of stimulation frequencies from single pulse to 20 Hz. Two-way ANOVA revealed that increasing stimulation frequency increased dopamine release in all photoperiod groups, as expected  = 13.18, p < 0.0001; F = 10.43, p < 0.0001). Specifically, Tukey’s post hoc multiple comparisons test showed that dopamine release was elevated in NAc core from Long photoperiod mice compared with NAc core from Equinox photoperiod mice in the 10-Hz (p = 0.018) and 20-Hz (p = 0.046) conditions. Taken together, our results indicate that Long summer-like photoperiods can enhance evoked dopamine release in the NAc core.We measured electrically evoked DA release from the NAc core in operiods A–C. The operiods D, shows operiods E. Peak dN = 4, females N = 5), Equinox , and Long  where N is number of animals. Using a two-way ANOVA, we found significant effect of stimulation frequency per se in both males  = 5.667, p = 0.002) and females  = 10.26, p < 0.0001). Strikingly, we found that Long photoperiod enhanced dopamine release only in females  = 26.69, p < 0.0001; F = 0.7842, p = 0.4622; post hoc tests showed significant differences in NAc core dopamine release between Long and Equinox photoperiod females at the single pulse (p = 0.029), 5-Hz (p = 0.002), 10-Hz (p = 0.0002), and 20-Hz (p = 0.001) stimulation frequencies, as well as significant differences between Short and Long photoperiod females at the 5-Hz (p = 0.031), 10-Hz (p = 0.003), and 20-Hz (p = 0.016) stimulation frequencies. Thus, the overall increase in NAc core dopamine release in Long photoperiod described above in As there are known sex differences in the regulation of mesolimbic dopamine , we invemaxV; F = 3.424, p = 0.047). Tukey’s post hoc multiple comparisons test showed that NAc core from Long photoperiod females had elevated dopamine release compared with those from Equinox photoperiod females (p = 0.02). We also found a significant main effect of photoperiod on dopamine uptake  = 6.029, p = 0.007) as well as a significant interaction of sex and photoperiod  = 4.334, p = 0.023) using two-way ANOVA. Maximal dopamine uptake in the NAc core was enhanced in Long photoperiod females compared with both Short (p = 0.004) and Equinox (p = 0.001) photoperiod groups as tested by Tukey’s post hoc multiple comparisons test. We did not observe any significant differences in dopamine release or uptake in the males as tested by Tukey’s post hoc multiple comparisons test. Taken together, our results indicate photoperiod has female-specific effects on both release and uptake of NAc core dopamine.To assess possible synaptic mechanisms through which photoperiod exerts its effects on NAc core dopamine, we analyzed concentration of single pulse evoked DA release in females and males E and formK across doses of GBR 12783 and photoperiods , Equinox , and Long  where N is number of animals. Consistent with F = 3.351, p = 0.0442; F = 1.596, p = 0.2153; mK of DAT in response to uptake inhibition showed main effects of photoperiod  = 16.21, p < 0.0001) and of GBR 12783 dose  = 12.27, p < 0.0001), as well as a significant interaction of photoperiod and dose  = 4.585, p = 0.0009; m compared with Short (p = 0.019) and Equinox females (p = 0.017) and at 3 μm compared with Short (p < 0.0001) and Equinox females (p < 0.0001) using Tukey’s post hoc multiple comparisons test. Thus, pharmacological inhibition of DAT by GBR 12783 was more sensitive in the NAc core of Long photoperiod female mice. We did not find a significant effect of photoperiod on pharmacological DAT inhibition in males  = 0.3393, p = 0.7143; As photoperiod may exert its effects on NAc core dopamine release and uptake in females through modulation of DAT, we examined a role for DAT using a selective DAT inhibitor (GBR 12783). We measured peak dopamine release and uptake and derived changes to apparent DAT operiods . Sample Here, we have laid the groundwork for the broader question of how environmental signals can program or shape the neural circuits that promote affective behavior. We have shown that photoperiod, one of the most pervasive and reliable environmental signals, impacts mesolimbic dopamine signaling, a key component of the neural circuitry that contributes to affective state. Our results, for the first time, demonstrate that photoperiod fine-tunes the subsecond signaling dynamics of neuromodulatory synapses.Photoperiod-dependent changes in dopamine were only detected in females, where Long summer-like photoperiods enhanced dopamine release and uptake. This female-specific photoperiod effect was present at all stimulation frequencies we tested. This indicates that the enhancement of dopamine release in Long photoperiod females was not dependent on stimulation frequency, suggesting that Long-summer like photoperiod enhances dopamine release in both tonic and phasic firing conditions. There are several possible mechanisms through which this could occur. Cholinergic interneurons (CINs) in the NAc can enhance and even directly elicit dopamine release from terminals in the NAc through presynaptic nicotinic acetylcholine receptors . There iWe also found that dopamine uptake was enhanced in the Long-photoperiod females. Because DAT activity can influence dopamine release through the recycling of transmitter , it is aPrevious studies have shown that increased DAT expression in genetic and experience-dependent models in rodents does not affect the potency of DAT inhibitors , 2014. RThe sex-specific effects of photoperiod we have observed here warrant further investigation of the mechanisms through which photoperiod affects males and females differently. There are at least three major biological phenomena that can underlie sexually dimorphic phenotypes: sex hormone action in the adult organism, sex hormone action during development, and genetic influence of sex chromosomes . EstrousWe were intrigued to find that dopamine release and uptake in the NAc core of male mice were not significantly influenced by photoperiod, especially since previous work using males have shown photoperiod can impact the striatal dopamine system by several measures. Findings from +/+ strain. The commonly used C57/BL6J strain (C57), does not produce physiological levels of endogenous melatonin because of genetic mutations in the melatonin synthesis pathway (+/+ mice depends on melatonin signaling through the melatonin MT1 receptor (rd gene that results in rod/cone degeneration rendering them visually impaired (+/+ strain has had the retinal degeneration mutation bred out (Another important distinction between previous photoperiod studies and our own is our use of the melatonin-competent C3Hf pathway . Melaton pathway . Interesreceptor . The spereceptor . This stimpaired , but as impaired . The C3Hbred out  so that Several studies have shown that nearly all aspects of dopamine transmission, including synaptic dopamine release, DAT activity, expression of dopamine receptors, dopamine synthesis and dopamine-dependent behaviors show diurnal rhythms . ExperimPrevious work from our group used the same mouse model and photoperiod conditions to test how seasonally relevant photoperiods impact affective behaviors . Mice raNpas2, has female-specific effects on vulnerability to substance use (Npas2 mutant females, especially in the dark phase, showed increased cocaine self-administration, faster acquisition of cocaine self-administration, higher break point ratio, and increased extinction responding (Npas2 mutant females, suggesting that ovarian hormones mediate these female-specific, circadian-dependent effects (Cocaine-related behaviors in rodents have also been shown to be circadian , and disance use . Npas2 msponding . These e effects . Because effects . HoweverThe mesolimbic dopamine system is implicated in the pathophysiology of many neuropsychiatric disorders, including major depressive disorder MDD; , seasonaTaken together, our study demonstrates that photoperiod impacts the subsecond release and uptake dynamics of synaptic mesolimbic dopamine, revealing a novel process by which photoperiod impacts neural systems involved in reward and affective behaviors. These findings identify a potential role for the dopamine system in mediating sex differences in seasonality on a synaptic basis, and an animal model in which to further explore potential mechanisms of sex differences in neuropsychiatric disorders.


Molecular simulation is becoming a standard tool for researchers working in different fields, such as physics, chemistry, material science, biology, medicine, engineering, and many others. Simulations provide information that cannot be obtained from experiments and offer scientists new cognitive opportunities. Advanced computational methods are used for a rational design of new chemical compounds, macromolecules, nanoparticles, and more complex assembled structures at any level of organization, from molecular to viruses.A major goal of this Special Issue is to show a wide range of applications of molecular simulations and to draw together contributors from different disciplines to stimulate the exchange of knowledge and experiences. The collection of articles gathered here can be divided into two groups. The first group concerns the modeling of the internal structure of macromolecules and interactions between different complex chemical structures. The second represents typical physico-chemical studies systems containing many molecules.Most works belong to the first group, which to some extent reflects the general trend in molecular sciences. The research is focused on specific chemical compounds with potential applications in medicine. In particular, the studies were aimed at improving the treatment of serious diseases, especially cancer, and preventing antibiotic resistance. Other articles are devoted to pesticide resistance and biomembrane modeling.Tran et al. describe the effects of alternative one-point mutations on the New Delhi Metallo Biomembranes consist of lipids, proteins, and carbohydrates and are fundamental to life. They are responsible for the cell envelopes that enable cells to absorb nutrients and exclude the most harmful agents from entering cells. The protein-lipid interactions are crucial for these processes. The work by Chen et al. concerns the archaeal lipids regulating the trimeric structure dynamics of bacteriorhodopsin for efficient proton release and uptake. They carried out long-term all-atom molecular dynamics simulation with the single-point mutagenesis to delineate the molecular mechanism of the specific interaction of the considered archeal lipids with bacteriorhodopsin during the proton release and uptake processes and found that these lipids are essential for stabilizing the bacteriorhodopsin trimer and maintaining the coherent conformational dynamics necessary for proton transfer.The second group of works represents typical physico-chemical studies of systems consisting of a great number of particles. These articles concern simulations of the following phenomena: (i) adsorption on special substrates, (ii) phase transitions, and (iii) self-assembly.Raza and co-workers performed molecular dynamics simulations to study the adsorption behavior of hydrogen, carbon dioxide, and methane on four sub-models of kerogens of varying thermal maturities over a wide range of pressures and temperatures. These simulations have a purely practical aspect namely they show that the organic-rich shales can be used for competitive Phase transitions are the subject of two articles. Mareev and Potemkin present the molecular dynamics study of the morphology of silicon. They demonstrate an ultrafast reversible phase transition in silicon  nanoparticles which are perceived as high-performance materials that combine the special features of cores and attached ligands. One work, however, refers to the formation of virus-like particles.Borówko and Staszewski investigate the polymer-tethered nanoparticles in narrow slit-like pores using coarse-grained molecular dynamics simulations. The confinement in pores creates new possibilities for controlling the shape transformation of individual hairy particles and their self-organization. The impact of interactions with the surfaces and the wall separation on the system morphology is discussed. Different ordered structures, resembling two-dimensional crystalline lattices, are reported. The paper by Sato et al. provides fundamental insight into the sensing applications of polymer-tethered nanoparticles. They use the dissipative particle dynamics method to investigate the influence of selected parameters on sandwiching properties of Janus-type tethered nanoparticles. Nanosensors are beneficial devices that can convert microscopic information derived from the behavior of atoms and molecules into macroscopic data. For example, biomolecules can be sandwiched between nanoparticles and detected by surface-enhanced Raman scattering. Controlling the gap between extremely close nanoparticles and stably capturing the target molecule in the gap are crucial aspects of this strategy.Macromolecular self-assembly is the basis of many phenomena in life and material sciences that find diverse applications in technology. A prominent example is the formation of virus-like particles that act as stable empty capsids used for drug delivery or vaccine fabrication. The virus-like particles are, in turn, protein assemblies. Depta et al. present the data-driven modeling approach for capturing macromolecular self-assembly on scales beyond traditional molecular dynamics while preserving the chemical specificity. Each macromolecule is represented by an anisotropic object and high-dimensional models are formulated to describe interactions between molecules and with the solvent. The self-assembly process is modeled as a combination of diffusive effects and pairwise interaction of molecules. The Kriging-based strategy building upon high-throughput molecular dynamics simulations with the Martini force-field is employed including semi-automated supervised learning to derive data-driven protein–protein interaction potentials. This generic method is applied to capture the formation of hepatitis B virus-like particles from the smallest building unit (the dimer of the core protein HBcAg). Assembly pathways and kinetics are analyzed and compared to the available experimental observations.Taken together, all articles published reflect well the most interesting current directions in molecular simulation studies. It is the pleasure of the Guest Editor to thank all authors for their important contributions to this Special Issue.


Vibrio vulnificus infections develop rapidly and are associated with a high mortality rate. The rates of diagnosis and treatment are directly associated with mortality.V. vulnificus infection in winter. Within 12 h of arrival, typical skin lesions appeared, and the patient rapidly developed primary sepsis. Despite prompt appropriate antibiotic and surgical treatment, the patient died 16 days after admission.We describe an unusual case of a 61-year-old male patient with chronic liver disease and diabetes who presented with a chief complaint of pain in both lower legs due to V. vulnificus infection should be suspected in patients with an unclear infection status experiencing pain of unknown origin in the lower legs, particularly in patients with liver disease or diabetes, immunocompromised status, and alcoholism.Our case findings suggest that  Vibrio vulnificus is a gram-negative, halophilic, alkaliphilic marine bacterial pathogen commonly found in plankton and shellfish, especially oysters, which was first described in 1976. [V. vulnificus favorably grows in warm water and low-salinity areas in coastal regions; therefore, V. vulnificus infections show seasonal and regional patterns [V. vulnificus infections are generally acquired by consuming contaminated seafood, particularly oysters. Wound infections can occur after skin lesions are exposed to contaminated seawater [in 1976. . V. vulnpatterns . V. vulnseawater , 5.V. vulnificus infection in winter in our hospital located in Binzhou, a coastal city in eastern China. The patient complained of pain in both lower legs without presenting with other prodromal symptoms upon admission. To the best of our knowledge, this clinical presentation is unusual and could alert clinicians to possible V. vulnificus infection in patients with an unclear infection status, who experience pain of unknown origin in the lower legs even before the appearance of typical skin lesions, especially if the patients have risk factors.In this report, we present an unusual case of necrotizing fasciitis with sepsis presenting with pain in the lower legs caused by A 61-year-old male patient was transferred to our hospital on November 29, 2020, at 23:30 pm, with complaints of pain in both lower legs for 16 h. He had a medical history of hepatitis B, hepatitis B-decompensated cirrhosis, and liver cancer, along with hypertension, diabetes mellitus, and tobacco use (2 packs of cigarettes per day). Moreover, he had a history of esophageal variceal ligation under endoscopic surgery 5 years earlier. He had consumed seafood  for lunch 3 days before admission.The patient was in a normal state until the morning of the 3rd day after consuming seafood with his family when he developed pain in both lower legs on awakening. He could recall no traumatic events or any history of recreational marine exposure. He did not consider the pain as a concern and ingested 800 mg of ibuprofen and sprayed diclofenac sodium on his lower legs. He experienced worsening of pain and arrived at our hospital at night. Color ultrasound examination of the legs showed normal findings.Initial physical examination revealed the following: clear consciousness; body temperature, 36.5 °C; respiration rate, 17 breaths per minute; blood pressure, 92/50 mmHg; and heart rate, 68 beats per minute. Reddish patches were observed on the right ankle.9/L ; platelet count, 35 × 109/L; hematocrit, 28%; serum glucose, 8.73 mmol/L; alanine aminotransferase, 22.84 U/L; ceramic oxaloacetic transaminase, 34.3 U/L; total bilirubin, 32.7 µmol/L , serum γ-glutamyltransferase, 101.4 µmol/L; prothrombin time, 18.8 s; partial thromboplastin time, 43.5 s; internationally standardized ratio, 1.64; d-dimer, 3.07 mg/L; serum urea nitrogen, 14.88 mmol/L; myoglobin, 672.2 ng/mL; creatine kinase, 210.2 U/L; C-reactive protein, 223.90 mg/L; blood sodium, 124 mmol/L; blood chlorine, 93.90 mmol/L; and serum potassium, 5.52 mmol/L. Hemoglobin, hematocrit, creatinine, and routine urine test results were normal.The initial laboratory investigations revealed the following: white blood cell count, 6.1 × 10Blood test results indicated thrombocytopenia, unclear infection status, coagulation dysfunction, hypoalbuminemia, electrolyte disturbance, and renal dysfunction. Blood culture was performed, and empirical antibiotic therapy was initiated. We used levofloxacin  intravenously.9/L. Color ultrasound and magnetic resonance imaging of the legs revealed extensive swelling in the soft tissue of the lower legs, suggesting cellulitis and edema , and he was immediately admitted to the intensive care unit. Intravenous therapy with imipenem/cilastatin sodium , cefoperazone sodium/sulbactam sodium , and levofloxacin was initiated. Four days after admission, bacterial cultures from the blood, bullae fluid, and tissues (matrix-assisted laser desorption/ionization–time of flight mass spectrometry) were all positive for Despite prompt appropriate antibiotic and surgical treatment, the skin lesions spread to the right thigh on the 2nd day after surgery Fig. . AmputatVibrio vulnificus multiplies in approximately 10 min at 20 °C, and its multiplication is controlled below 15 °C [V. vulnificus infections are common in warm climates, but our case occurred in winter. Although it is relatively unusual, there have been previous reports of cases in cold climates [ow 15 °C . V. vulnclimates ; thus, wV. vulnificus infection, particularly those with hepatic cirrhosis. This may be due to the fact that patients with chronic liver disease often have portal hypertension, which can cause shunting of the bacteria around reticuloendothelial cells in the liver [V. vulnificus more likely to enter the bloodstream and cause bacteremia on consumption of contaminated seafood. V. vulnificus disseminates from the gastrointestinal tract to the bloodstream, leading to the rapid development of primary sepsis. This may explain why only our patient developed primary sepsis 3 days after consuming seafood, although all his family members consumed the same diet.Previous reports have shown that patients with liver disease are more susceptible to he liver . Other rhe liver , 9, 10. he liver . The patV. vulnificus can progress to necrotizing soft-tissue infections [V. vulnificus (contact with seawater or raw seafood) or Aeromonas species  [Infection with this pathogen can result in three major discernible manifestations: primary sepsis, wound infections, and gastrointestinal diseases , 12. Prifections . The conor wood) . In addid-dimer, and coagulation dysfunction may indicate progression to disseminated intravascular coagulation. Moreover, elevated creatine kinase may signify rhabdomyolysis. We should obtain critical information quickly when treating patients with suspected limb infections, which can help identify patients with life-threatening limb infections at an early stage.In the present case, the patient presented with pain in both lower legs as the chief complaint. We did not detect any other prodromal symptoms; therefore, diagnosis was challenging until the development of the characteristic bullae. Despite the inapparent or atypical presentation, there were some indicators of infection. His blood pressure was slightly low, and laboratory examination findings suggested infection; these may indicate septic shock. Reddish patches on the right ankle, elevated V. vulnificus infection. A recent study suggested that for patients presenting with septic shock with a recent history of raw seafood consumption, treatment with doxycycline or ciprofloxacin for appropriate coverage of V. vulnificus and gram-negative resistant organisms should commence while awaiting microbiological cultures. Once a diagnosis of V. vulnificus septicemia is confirmed, treatment can be safely changed to ceftriaxone combined with doxycycline or ciprofloxacin [V. vulnificus infection [Antibiotic therapy and debridement are the most effective methods for the treatment of necrotizing myofascitis caused by floxacin . Early dnfection . In the V. vulnificus is a deadly and opportunistic human pathogen that usually infects humans through seafood consumption or direct contact with open wounds. Infection with this pathogen can rapidly progress to septic shock and even death. Therefore, early culture and diagnosis are crucial. Our case alerts clinicians to a possible V. vulnificus infection in patients with an unclear infection status, who experience pain of unknown origin in the lower legs, especially if the patient has hypotensive shock, leukopenia, hypoalbuminemia, coagulation dysfunction, increased creatine kinase, or underlying liver disease and diabetes.In conclusion,