We aimed to examine clinical data and baseline blood test results as potential predictive biomarkers for benefit from nivolumab, in advanced non-small cell lung cancer patients (NSCLC).A chart review was performed of 108 advanced NSCLC patients who commenced treatment with nivolumab between 2015-6 at three Israeli cancer centers, and for whom laboratory tests results were available. Data collected included sex, age, ECOG-PS, histology and number of previous lines of treatment. Baseline blood test results collected: absolute lymphocyte and neutrophil count (ANC), white blood cells (WBC), hemoglobin, platelets, albumin and lactate dehydrogenase (LDH). Neutrophil to Lymphocyte Ratio and ‘derived NLR’ (dNLR = (ANC/[WBC-ANC])) were calculated. Disease control at six months (DC6) was defined as any tumor shrinkage or stable disease during the first six months of nivolumab treatment. The association between clinical/laboratory variables and survival was tested with a Cox proportional hazard model. Data cut-off occurred in November 2019.35 patients (32.4%) achieved DC6. Median overall survival (OS) of entire study population was 5.4 months. Four year survival rate was 16%. Achievement of DC6 strongly correlated with longer OS . In univariate and multivariate analysis, dNLR, albumin and LDH correlated significantly with OS. No variables correlated significantly with DC6 in multivariate analysis. Based on albumin and LDH, we produced a score called CLAS , including four prognostic groups of patients. Patients having low albumin and high LDH had the worst prognosis.In real-life setting, long-term efficacy of nivolumab in advanced line treatment of NSCLC is consistent with clinical trials. Response or stability of disease during first six months of treatment is associated with prolonged survival. We propose a novel score (CLAS) that may be useful for predicting outcome in nivolumab-treated NSCLC patients, but further validation is required. Atezolizumab is similarly approved by FDA and EMA, while pembrolizumab is approved only for the treatment of patients with PD-L1 positive tumors   in the tumor .Levels of peripheral blood components, blood cells and various circulating molecules are an accessible potential non-invasive source of predictive biomarkers in NSCLC patients, and possibly relevant also for ICI-treated patients. Some of these biomarkers are validated prognostic markers for cancer in various scenarios. For example, high levels of serum lactate dehydrogenase (LDH) has been associated with poorer cancer-specific survival in several malignancies, including lung, colorectal and prostate cancer . AnotherA number of studies investigated the peripheral blood cellular components as biomarkers of ICI efficacy. For ipilimumab-treated melanoma patients, improved OS and PFS were associated with low absolute neutrophil count, low neutrophil-to-lymphocyte ratio and high lymphocyte levels , 29. In versus chemotherapy for NSCLC patients and compiled a Lung Immune Prognostic Index (LIPI)  . In thisx (LIPI) , 36. In x (LIPI) .We report here real-world long-term survival results of NSCLC patients treated with nivolumab as a second or later treatment line in three Oncology centers, with a median follow-up of four years. We have comprehensively assessed clinical data and baseline blood levels of LDH, albumin and complete blood count results, as potential predictive biomarkers for benefit from nivolumab.This is a retrospective pharmacoepidemiological study, conducted, analyzed and reported according to REporting of studies Conducted using Observational Routinely collected health Data (RECORD) guidelines, the Strengthening the Reporting of OBservational studies in Epidemiology (STROBE), and ROCORD-pharmacoepidemiological research (RECORD-PE) guidelines –40. A chInclusion criteria were age of 18 years and above, progression on first-line platinum-based chemotherapy, treatment at one of three participating Israeli cancer centers , Rabin MC and Shamir MC), administration of at least one cycle of nivolumab, with treatment commencing during 2015-2016. Exclusion criteria was lack of blood test results from the relevant time window , histology and number of previous lines of treatment. Baseline blood test results collected included: absolute lymphocyte count (ALC), absolute neutrophil count (ANC), white blood cells (WBC), hemoglobin (HGB), platelets (PLT), albumin (ALB) and LDH. Baseline blood tests were defined as those performed prior to and within two weeks of the first nivolumab treatment. These parameters were categorized as high or low relative to the median of the study cohort, except for LDH, categorized as normal or above the upper limit of normal for the relevant clinical laboratory and albumin, categorized as normal or below the lower limit of normal for the relevant clinical laboratory. LIPI calculation is based on LDH greater than upper limit of normal (ULN) and ‘derived NLR’ ) > 3. Three risk groups were characterized: good: none of these factors, intermediate: one factor, poor: two factors. NLR, dNLR and LIPI score were calculated. In addition, CLAS (“Combined LDH and Albumin Score”) was defined as a suggested novel score and calculated as described in the results section.Tumor assessments were performed by the treating physicians based on computerized tomography (CT) scans performed as part of the standard of care. CT scans were usually performed at two-three months intervals, at the treating physicians’ discretion. Two major outcomes have been assessed in our study: Disease control at six months (DC6) and OS. DC6 was defined as either present  or absent (any tumor growth or death at any time within the first six months).OS was defined as time from initiation of nivolumab treatment until death, or censored at the last date patient was known to be alive.Variables analyzed as categorial included sex and histology. In addition, LDH was redefined as a binary variable based upon whether the values being above or below the upper-limit of normal, and ALB was redefined as a binary variable based upon whether the values were above or below the lower-limit of normal. Variables analyzed as continuous included age, HGB, WBC, ANC, ALC and PLT. Variables analyzed as ordinal were ECOG-PS and number of previous lines.2 values. Calculations were performed using Stata . Data cut-off was at November 2019.The impact of covariates on survival was assessed using a Cox proportional hazard model. Multivariate analysis incorporated putative covariates found at univariate analysis to be significant at p < 0.1. Statistical comparisons were performed using Chi-squared test for categorical data and t-test for continuous variables. All p-values were two-sided, and p < 0.05 was considered statistically significant. The goodness-of-fit of different models were compared by examining pseudo-RThe study was approved by the institutional ethics committees of each of the participating centers. : IRB #8993-11; Rabin Medical Center (MC): IRB #0391-14; Shamir MC: IRB #0062-17).Investigators had complete access to the database of population included in the study. No patient identifying information was included in the study database.A total of 108 patients treated with nivolumab were included in this study, mostly men (65%). Out of the study cohort, 35 patients (32.4%) had achieved DC6. More patients in the non-DC6 group had ECOG-PS of two or higher compared with DC group (p=0.030). The two groups were balanced in terms of other parameters Table 1.The median OS of the entire study population was 5.4 months  0.07-0.21, p<0.001; We next tested the effects of the variables collected  on OS. On univariate analysis, ECOG-PS and baseline values of WBC, ANC, NLR, dNLR, LDH and albumin were significantly correlated with OS. Due to overlap with dNLR, the variables WBC, ANC and NLR were not included in the multivariate analyses. On multivariate analysis, dNLR, albumin and LDH significantly correlated with OS , leading to median OS for poor, intermediate and good LIPI of 2.1 months, 6.4 months, and 9.8 months, respectively Figure 4Of the parameters examined in our study, baseline LDH and albumin levels were found to be most significantly correlated with survival. We attempted to produce a predictive score by combining these two variables, naming it ‘CLAS’. CLAS includes four prognostic groups of patients: high LDH + low albumin (CLAS 0), low LDH + low albumin (CLAS 1), high LDH + high albumin (CLAS 2), low LDH + high albumin (CLAS 3). Indeed, patients classified as CLAS 0 had the worst survival (p<0.001), while patients with CLAS 2 had better survival than patients with CLAS 1, suggesting that low albumin is worse than high LDH  follow up of our cohort, we report now real-world data of survival of a set of NSCLC patients treated with 2ectively , as may ectively . We alsond treatment line. It should be noted that despite these poor prognostic characteristics, the long-term survival curve looks similar, with 16% long term survivors at data cut-off, consistent with data from the previously mentioned checkmate trials (The relatively shorter median OS in our study may be attributed to the inclusion of patients with ECOG-PS 2/3 that constituted 52% (57 patients) of our cohort, while the randomized studies included only ECOG-PS 0/1 patients. Furthermore, 16% of the patients in our study were heavily pretreated, receiving nivolumab as a later than 2e trials .versus being general prognostic biomarkers.The two parameters outstanding from our data, which were included in the suggested ‘CLAS’ reflect tumor burden (LDH) and the patients’ nutritional status (ALB). The three variables that make up CLAS and LIPI score  surprisingly performed better than ECOG-PS as predictors of OS. Despite the recognized validity of ECOG-PS as a strong prognostic factor, the inter-observer variability of this parameter may limit its usefulness in some cases . An impoA limitation of the study is its retrospective nature, with recognized drawbacks in terms of data accuracy and non-regular follow-up intervals. Furthermore, radiological response to treatment was based on investigators’ assessment and not on RECIST. In addition, the relevance of this cohort can be questioned, as most patients are now treated front-line with ICI (either as monotherapy or combined with chemotherapy). We raise here the possibility CLAS score could be applicable in the first-line setting as well, and examining our score in this setting is justified.Another limitation is the relatively small size of the study, and the lack of a validation cohort to assess the accuracy of the suggested CLAS classifier. Such a validation cohort with equivalent prolonged follow up will be available in the future. However, our data of correlates of long-term survival allows insight into factors correlating with significant ICI efficacy, potentially with the postulated chance of cure. A larger data set is required for comparing the utility of LIPI and CLAS.In conclusion, unlike median OS, the real life long-term efficacy of nivolumab in the advanced line setting in NSCLC is similar to data from published randomized trials. In addition, we propose a novel score we named ‘CLAS’ based on baseline albumin and LDH results as a potentially useful score for predicting outcome in nivolumab-treated NSCLC patients. The simple and unbiased measurement of these values adds to their apparent clinical applicability. This score needs further validation before any practical conclusions can be reached. Furthermore, we suggest categorization of patients on immunotherapy according to disease control after six months of treatment to be a simple and useful tool for similar studies. DC6 reflects the survival benefit of responding patients as well as of those with prolonged stability, and marks them as having a good chance for durable response and prolonged survival. Thus, this parameter should be further investigated as a surrogate factor for OS in ICI studies.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The study was approved by the institutional ethics committees of each of the participating centers. . The studies involving human participants were reviewed and approved by Sheba Medical Center ethics committee. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.SD: Conceptualization, Methodology, Investigation, Writing - original draft. YL: Formal analysis, Data curation, Writing - Review & Editing. ED: Conceptualization, Methodology, Investigation, Writing - Review & Editing. EH: Investigation, Writing - Review & Editing. DU: Conceptualization, Methodology, Writing - Review & Editing. NP: Conceptualization, Methodology, Writing - Review & Editing. RN: Resources, Writing - Review & Editing. RL: Conceptualization, Methodology, Writing - Review & Editing. AH: Resources, Data curation, Writing - Review & Editing. EB: Resources, Data curation, Writing - Review & Editing. TG: Resources, Formal analysis, Writing - Review & Editing. AO: Conceptualization, Methodology, Writing - Review & Editing. JB: Conceptualization, Methodology, Validation, Supervision, Project administration, Writing - Review & Editing. All authors contributed to the article and approved the submitted version.Nivolumab was provided to the majority of patients by Bristol-Myers Squibb (BMS) as part of a compassionate use program. BMS was not involved in collection, analysis, interpretation of data, manuscript drafting or submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The boll weevil is a destructive pest of commercial cotton throughout the Americas. An eradication program in the United States has removed the boll weevil from most of its range. However, weevil populations in South Texas remain a threat to eradicated areas. Pheromone traps are used to monitor boll weevil activity, and when a weevil is captured, eradication programs rely on malathion for control. However, the effectiveness of pheromone traps in detecting incipient boll weevil populations is reduced during certain times of the year. Additionally, human safety and environmental concerns, as well as the potential development of malathion-resistant populations, have prompted program managers to seek alternative control methods. We sequenced and compared pheromone-producing and non-pheromone-producing weevils to identify genes involved in pheromone production, which, in turn, could be an environmentally friendly way to target gene-level pest control that is specific to the boll weevil. Our results revealed genes involved in pheromone production, as well as insect development and immunity, which may be targeted for boll weevil suppression.Anthonomus grandis grandis Boheman (Coleoptera: Curculionidae), rely almost exclusively on pheromone traps to indicate the need for insecticide applications. However, the effectiveness of traps in detecting weevil populations is reduced during certain times of the year, particularly when cotton is actively fruiting. Consequently, this could result in fields becoming heavily infested with weevils. It is widely speculated that the lack of weevil captures in traps during this period is largely due to the overwhelming amount of pheromone released by weevils in the field, which outcompete the pheromone released from traps. Thus, this work sought to identify genes involved in pheromone production so that new control methods that target these genes can be explored. We conducted an RNA-seq experiment that revealed 2479 differentially expressed genes between pheromone-producing and non-pheromone-producing boll weevils. Of those genes, 1234 were up-regulated, and 1515 were down-regulated, and most had gene annotations associated with pheromone production, development, or immunity. This work advances our understanding of boll weevil pheromone production and brings us one step closer to developing gene-level control strategies for this cotton pest.Eradication programs for the boll weevil,  Anthonomus grandis grandis Boheman (Coleoptera: Curculionidae), is a pest of commercial cotton (Gossypium hirsutum) throughout the Americas [https://www.txbollweevil.org.html accessed on 19 September 2021). In fact, recent infestations in eradicated areas in Texas have resulted in multi-year infestations, which have cost millions of dollars to remediate .The boll weevil, Americas . EffortsAmericas ,3,4. How1,β-cyclohexaneethanol, respectively) and two terpene aldehydes  [Eradication programs rely exclusively on pheromone traps to detect and monitor boll weevil populations . Pheromoldehyde) . The pheldehyde) . During It is generally recognized that weevil captures in traps vary seasonally and decline rapidly once cotton begins to produce fruiting structures ,9,10. InOne way to avoid trap competition in the field is to develop a gene-based control strategy that suppresses the production of pheromone in the male weevil. This could increase the attractiveness of the pheromone lure in traps allowing for more reliable and early detection of weevils; thus, leading to more timely insecticide applications. Suppression of pheromone-producing weevils could also reduce and/or delay early-season colonization of cotton fields by overwintered weevils. Such delays or reductions could potentially prevent fields from becoming heavily infested , which iBoll weevils were initially collected as larvae in infested squares from a field northwest of Edinburg, TX . The major life stages  were placed in RNAlater , and stored at −20 °C until total RNA was extracted for the transcriptome assembly. A group of pupae from the same field was kept on vermiculite in a 29.4 ± 1 °C degree incubator to obtain adults for the pheromone experiment. Newly eclosed adults (<24 h old) were sexed, and only the males were kept. Forty males were divided into two groups. One group was held without food for 24 h. The other group was fed squares  daily for 6–8 days and provided water on a section of cotton dental wick saturated with deionized water to promote the production of all four pheromone components ,20. WeevRNA was extracted from weevils in each life stage using the RNeasy extraction kit . Samples were checked for quantity and quality using a Tapestation 4200 . The total RNA from 12 samples at different life stages  and 20 weevils from the pheromone experiment  were submitted to Texas A&M AgriLife Genomics and Bioinformatics Service (TxGen) for purification, library preparation, and sequencing on an Illumina NovaSeq , paired-end with a read length of 2 × 150 base pairs.de novo transcriptome using the TRINITY 2.12.0 assembler [−5.A total of approximately 30 million reads per sample were downloaded from TxGen and trimmed of adaptors and low-quality sequences using Trimmomatic 0.38 . Approxissembler . The defssembler . Completssembler ,26,27; sssembler . The trade novo transcriptome using RNA-seq by Expectation-Maximization (RSEM). The workflow also included quantifying the mapped reads at each locus and statistically assessing fold change  < 0.05) differences between pheromone-producing and non-pheromone-producing weevils [A total of approximately 30 million reads per sample  for the pheromone and non-pheromone samples were used in the RNA-seq analysis, which was accomplished using edgeR as part of the OmicsBox software [The final compiled transcriptome had 195,128 total transcripts with an average length of 922.89 bp, Dendroctonus ponderosae Hopkins and Sitophilus oryzae Linnaeus, followed by the tenebrionid, Tribolium castaneum Herbst. Nearly all sequences in the dataset aligned to other coleopterans, The BLAST annotation revealed most sequences aligned to two weevil species, Gene Ontology (GO) was performed for all three major categories: molecular function (MF), biological process (BP), and cellular component (CC). Standard gene groups were represented in all three categories, RNA-seq analysis identified a total of 2479 DEGs between the males that produced pheromone compared to those that did not produce pheromone. Of those 2479 genes, 1234 genes were up-regulated, and 1515 genes were down-regulated, Evidence from other coleopterans shows pheromone is made by utilizing the first section of the juvenile hormone pathway, called the mevalonate pathway, which uses byproducts to produce the monoterpenoid pheromone component . The remhydroxymethylglutaryl-CoA synthase 1 (HMG-S), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-R), and phosphomevalonate kinase were all highly up-regulated and found in the first steps of the mevalonate pathway converting acetyl-CoA to mevalonate 5-PP, the final step before isopentenyl synthesis, ATP-citrate synthase, which converts citrate to acetyl-CoA, and is needed to initiate the mevalonate pathway, The mevalonate pathway in insects is a metabolic pathway initiated by reductive polymerization of acetyl-CoA and leads to a diversity of isoprenoid compounds . Informadolichyl pyrophosphate. Similar molecules have also been shown to be part of the production of isoprenoid compounds in blowfly larvae [fatty acid synthase, and glycerol-3-phosphate phosphatase, and most likely function to increase the size of the fat body and hormone production. The increase in a particular type of fat body is a feature that correlates with reproductive maturity in the boll weevil [I. pini males and are speculated to regulate the mevalonate pathway [Our study found up-regulation in genes in the isopentenyl, dolicol, and farnesyl portions of the pathway as well, y larvae . Severall weevil . Furtherl weevil . There w pathway .shuttle craft-like and regucalcin-like, shuttle craft has mostly been studied in its role in the embryonic central nervous system development in D. melanogaster [shuttle craft regulates genes that are either involved in axon growth or the secretion of molecules needed for chemoattraction [regucalcin-like has been studied very little in insects but has been characterized in mammals as a calcium-binding protein that regulates many cell functions needed for hormonal stimulation [We also identified two genes that may function to stimulate hormone production in pheromone-producing male boll weevils, nogaster , for whitraction . Interesmulation . Thus, bT. castaneum that was highly induced in response to feeding [transmembrane protease serine 9-like is also involved in digestion in the boll weevil. We found six other DE serine peptidase sequences, outside of the top 50 gene list, that had A. grandis as a top BLAST hit and were previously identified as midgut serine proteases [Agser2p and Agser5p, were induced in response to feeding. The up-regulation of digestive peptidase genes in our dataset is fitting because pheromone-producing males were fed fresh squares daily whereas, the non-pheromone-producing males were not given food. The remaining up-regulated serine peptidase genes could be involved in digestion as well but may also have roles in development and apoptosis, both of which may be necessary for boll weevil sexual maturity and pheromone production.Aside from genes directly involved in pheromone production, we also found genes up-regulated in associated processes, such as digestion and immunity. The most highly up-regulated gene in the dataset was a serine protease, transmembrane protease serine 9-like,  feeding . Thus, iroteases . In thatDNA/RNA non-specific nuclease 1, was shown to be in the midgut of Bombyx mori and interferes with the RNA interference response. Because of its propensity to degrade dsRNA, it has been suggested the enzyme might be involved in the innate immune response against invading nucleic acids, such as RNA viruses [Tenebrio molitor, showed males were more attractive to females if they were immunocompetent [Two immune genes were differentially expressed protein spaetzle 3 and DNA/RNA non-specific nuclease 1. In Drosophila, spaetzle is involved in embryonic development but also has a role in the innate immune response. Spaetzle proteins bind to Toll receptors and activate antimicrobial peptide gene expression . The sec viruses . The fun viruses . In factompetent . Thus, hT. castaneum. The gene laccase 2 was found to play a major role in cuticle tanning for all life stages except the egg stage [dopamine D2-like receptor and dopamine N-acetyltransferase, that may play a part in weevil cuticle tanning. Additionally, we found four cuticle genes, two chitin synthase genes, and many other cuticle genes outside of the top 50 DEGs, all down-regulated in pheromone-producing weevils, Many of the genes that were significantly down-regulated in pheromone-producing weevils had functions consistent with the cuticle and some were directly involved in cuticle tanning and sclerotization, gg stage . Likewisgg stage . We fountranscription factor ken, was also significantly down-regulated in pheromone-producing boll weevils, D. melanogaster, a transcription factor named ken and barbie, has a distinct role in the development of male and female external genitalia and is expressed in the highest amounts during embryo and pupal stages of development. In fact, mutants of this gene gave a range of deformities, including no external genitalia [The gene enitalia . This isde novo transcriptome with a BUSCO score of 99.27%. We used this assembly to perform an RNA-seq analysis comparing gene expression between pheromone-producing and non-pheromone-producing boll weevils. We ultimately identified significant DEGs that likely have key functions in pheromone production and biosynthesis. In subsequent experiments, successful knockdown of these DEGs should result in boll weevils with no or limited pheromone production, which could result in increased detection of weevils with traps. Because eradication programs in the U.S. rely almost exclusively on pheromone traps to indicate the need for insecticide applications, improved detection of boll weevils with traps could lead to more timely application of insecticides. Furthermore, disruption of boll weevil pheromone production may reduce early-season colonization of weevils that rely on pheromone to find cotton. Finally, the other DEGs identified in this study involved in immunity, cuticle formation, and digestion may also serve as targets for gene-level pest control. Thus, this RNA-seq analysis provides the foundation for the production of gene-level control strategies that may target pheromone production and other life functions in the boll weevil. Control strategies that incorporate these methods, such as RNA interference or CRISPR, would not only be specific to the boll weevil but would also be safe and environmentally friendly.We provide a boll weevil


Evaluating health-related quality of life (HRQOL) is important because it reflects the impact of treatment from the patient’s perspective. This holds especially true when prognosis is poor, as is the case for gastric cancer patients. This systematic review was performed to evaluate the short- and long-term impacts of current treatments on HRQOL in patients with locally advanced gastric cancer. The results of this review show that surgery and chemoradiotherapy have a significant impact on short-term HRQOL, although this recovers after 6–12 months. More focus on HRQOL in current clinical practice might improve patient counselling and monitoring before, during, and after treatment in locally advanced gastric cancer patients.Background: Current treatment strategies have been designed to improve survival in locally advanced gastric cancer patients. Besides its impact on survival, treatment also affects health-related quality of life (HRQOL), but an overview of reported studies is currently lacking. The aim of this systematic review was therefore to determine the short- and long-term impact of chemotherapy, surgery, and (chemo)radiotherapy on HRQOL in locally advanced, non-metastatic gastric cancer patients. Methods: A systematic review was performed including studies published between January 2000 and February 2021. We extracted studies published in Medline, Embase, and Scopus databases that assessed HRQOL in patients with locally advanced, non-metastatic gastric cancer treated with curative intent. Studies using non-validated HRQOL questionnaires were excluded. Short-term and long-term HRQOL were defined as HRQOL scores within and beyond 6 months after treatment, respectively. Results: Initially, we identified 8705 articles  and ultimately included 10 articles. Most studies reported that short-term HRQOL worsened in the follow-up period from 6 weeks to 3 months after surgery. However, recovery of HRQOL to preoperative levels occurred after 6 months. After completion of chemoradiotherapy, the same pattern was seen with worse HRQOL after treatment and a recovery of HRQOL after 6–12 months. Conclusions: In patients with locally advanced, non-metastatic gastric cancer, HRQOL deteriorated during the first 3 months after surgery and chemoradiotherapy. However, the long-term data showed a recovery of HRQOL after 6–12 months. To implement HRQOL in clinical decision making in current clinical practice, more research is needed. As the fourth cause of cancer-related mortality worldwide, gastric cancer remains a major health problem . DespiteThe aim of this systematic review is to provide an overview of the available literature on HRQOL in patients treated for locally advanced, non-metastatic gastric cancer. We have therefore formulated the following research questions: (1) What is the short-term impact of chemotherapy, surgery, and (chemo)radiotherapy on HRQOL in patients with locally advanced, non-metastatic gastric cancer? (2) What is the long-term impact of treatment on HRQOL in patients with locally advanced, non-metastatic gastric cancer?We performed a systematic literature review which is registered in the PROSPERO database, an international database of prospectively registered systematic reviews (registration number: CRD42021249987). We used the PRISMA  guidelines for our protocol and the systematic review .A systematic search of the Medline, Embase, and Scopus databases was performed on 12 February 2021. No additional studies have been included after this date. We searched for articles assessing HRQOL in patients with locally advanced, non-metastatic gastric cancer treated with curative intent. The full search strategy is described in the Studies were included if they met the following inclusion criteria: (I) patients with locally advanced gastric cancer (Ib-IVa gastric according to the 8th TNM staging of the American Joint Committee on Cancer) treated with curative intent adjuvant chemotherapy or adjuvant (chemo)radiotherapy); (II) the use of a validated HRQOL questionnaire; (III) article written in English language; and (IV) publishing date from the year 2000 and onwards.In addition, if studies analyzed different stages of gastric cancer, only the locally advanced groups were used in the analysis.Studies were excluded if: a non-validated questionnaire was used, when no original data was presented, reviews, study protocols, or abstracts with no full text available. Studies researching various stages of gastric cancer were excluded if the proportion of patients exceeded 20% stage Ia. This study investigated the impact of multimodal treatment in gastric cancer, whereas Ia is often treated less intensively.Primary outcomes of the study were: (1) short-term impact of treatment on HRQOL; (2) long-term impact of treatment on HRQOL. Based on available literature, short-term HRQOL was defined as HRQOL scores within 6 months after treatment . Long-teAll studies  identified by the search were independently analyzed by two observers (R.v.A. and K.v.d.S.). Conflicting decisions were resolved by discussion and if persistent, screened by a third reviewer (I.W.). R.v.A. and K.v.d.S. extracted the following data of the included studies: author, number of patients included, patient characteristics , study design, inclusion years, country, HRQOL questionnaires, time points of questionnaires, HRQOL subscales, and treatment strategy. Authors of articles who did not present all data in the original manuscript were contacted for additional results. Unfortunately, we did not receive sufficient data to perform a pooled analysis.Risk of Bias was assessed and discussed by two observers (R.v.A. and K.v.d.S.). The revised Cochrane risk of bias tool (RoB2) was used to assess randomized trials . Non-ranA flowchart of the included articles in this systematic review is displayed in Baseline demographics are described in The EORTC cancer-specific questionnaire (QLQ-C30) was used in the majority of the studies , followed by the QLQ-STO22 , EQ-5D , FACT-G and FACT-Ga , and PGSAS-45 .This section included eight studies that examined the impact of surgery on HRQOL in gastric cancer patients. A summary of all the outcomes is displayed in Short-term HRQOL was impaired from 6 weeks to 3 months after surgery as described by Avery et al., and Munene et al. ,30. In aThe long-term impact on HRQOL was investigated by Jakstaite et al., 6 to 18 months after gastric surgery. Almost half of the patients also received adjuvant chemotherapy. Patients aged 65 years and older had better global HRQOL scores and social functioning. No significant impact on HRQOL was seen based on gender, clinical stage, or adjuvant chemotherapy . Kinami The impact of the type of surgery on HRQOL yielded mixed results. Munene et al., concluded that there was no difference in HRQOL between patients who underwent a partial or total gastrectomy . Huang eBoth Brenkman et al., and Xia et al., evaluated HRQOL in gastric cancer patients compared to the general population or healthy controls ,36. BrenThe impact of chemoradiotherapy on HRQOL was investigated in the following studies and summarized in Short-term HRQOL was worse after chemoradiotherapy as described by Zygogianni et al., and Goody/Kassam et al. ,24,37. ZThe impact of chemotherapy on HRQOL in locally advanced gastric cancer was evaluated in the following studies .The short-term impact of chemotherapy on HRQOL was not evaluated in the included studies. The long-term impact on HRQOL was described by Jakstaite et al., and Brenkman et al. ,34. AdjuThe aim of this systematic review was to investigate the impact of chemotherapy, surgery, and (chemo)radiotherapy on short- and long-term HRQOL in locally advanced, non-metastatic gastric cancer patients. Our systematic review is the first systematic review investigating HRQOL in locally advanced gastric cancer patients. We found that HRQOL was impaired until 6 months of follow-up but from 6 months onwards, HRQOL recovered to pretreatment levels in most patients.The impact of treatment is reflected as an impaired short-term HRQOL. Within studies researching the impact of surgery, a decline in physical, mental, and functioning scores were seen from 6 weeks to 3 months after surgery. This pattern was also observed after chemoradiotherapy. However, the long-term impact (after 6 months) showed a recovery of HRQOL for both surgery and chemoradiotherapy ,24,25,30We observed that HRQOL in locally advanced gastric cancer patients was worse compared to healthy controls or the general population ,36. ThisAnother interesting finding of this systematic review was that HRQOL deteriorates when disease recurs and prior to death ,30. ThisIn this systematic review, the impact of the type of surgery on HRQOL is unclear, as contradictory conclusions were made. Two studies did not describe a difference between the type of surgery ,33. ThisThe impact of chemotherapy is also under debate as Jakstaite et al., did not find an impact of adjuvant chemotherapy on HRQOL . Howeverp = 0.020, ES = 0.25) and less dysphagia  compared to the chemoradiotherapy group [Current treatment usually consists of perioperative chemotherapy. However, prospective studies that investigate the impact of perioperative chemotherapy on HRQOL in patients with locally advanced gastric cancer are currently non-existent. Hence, there is a great need for more research on this topic. Within the CRITICS study, HRQOL has been investigated as well. The CRITICS study compared perioperative chemotherapy to preoperative chemotherapy, surgery, and postoperative chemoradiotherapy . The firpy group .Despite the small number of articles describing HRQOL in locally advanced gastric cancer, this study shows a trend in HRQOL similar to other cancers, e.g., esophageal cancer. Treatment of esophageal cancer  also resulted in a short-term deterioration of HRQOL, specified as four months from the start of therapy . SimilarThis systematic review has some limitations. First, there is a lack of high-quality studies investigating the quality of life in patients with locally advanced gastric cancer. Therefore, we only included a very small number of articles, hindering the possibility to perform a pooled analysis. We requested additional data, but unfortunately, we did not receive sufficient data to perform a pooled analysis. Second, there is also a difference in the quality of the included studies. Most studies are not randomized, and 4 of the 10 (40%) studies were scored as having “some concerns” in the Risk of Bias analysis ,32,33,35A strength of this study is the focus on patients with locally advanced gastric cancer treated with curative intent, which is, to our knowledge, the first systematic review investigating this. Furthermore, we performed an extensive systematic review, including only studies published from the year 2000 and onwards. In addition, we only included studies that assessed HRQOL with validated questionnaires.In conclusion, this is the first systematic review investigating HRQOL in locally advanced, non-metastatic gastric cancer patients. The impact that treatment has on HRQOL in locally advanced, non-metastatic gastric cancer worsened after surgery and chemoradiotherapy but recovered after 6–12 months. This dynamic course of HRQOL is of added value in current clinical practice, to clarify the patient’s expectations. More research is needed to implement HRQOL in clinical decision-making.


Bacterial infections constitute a threat to public health as antibiotics are becoming less effective due to the emergence of antimicrobial resistant strains and biofilm and persister formation. Antimicrobial peptides (AMPs) are considered excellent alternatives to antibiotics; however, they suffer from limitations related to their peptidic nature and possible toxicity. The present review critically evaluates the chemical characteristics and antibacterial effects of lipid and polymeric AMP delivery systems and coatings that offer the promise of enhancing the efficacy of AMPs, reducing their limitations and prolonging their half-life. Unfortunately, the antibacterial activities of these systems and coatings have mainly been evaluated in vitro against planktonic bacteria in less biologically relevant conditions, with only some studies focusing on the antibiofilm activities of the formulated AMPs and on the antibacterial effects in animal models. Further improvements of lipid and polymeric AMP delivery systems and coatings may involve the functionalization of these systems to better target the infections and an analysis of the antibacterial activities in biologically relevant environments. Based on the available data we proposed which polymeric AMP delivery system or coatings could be profitable for the treatment of the different hard-to-treat infections, such as bloodstream infections and catheter- or implant-related infections. Antibiotics are highly successful drugs that save the lives of millions of people yearly and are essential in many important medical procedures, such as transplantation, novel tumor treatments and complex surgical procedures, including implantation of artificial body parts. Unfortunately, a growing number of infections are hard-to-treat with current antibiotics, due to the emergence of antimicrobial resistant (AMR) strains  and/or bAntimicrobial peptides (AMPs) are promising candidates for the development of novel therapies to combat hard-to-treat infections ,6. AMPs Unfortunately, the use of AMPs in vivo is limited by several factors connected to their peptidic nature, including a low selectivity towards bacterial cells over mammalian cells, rapid removal from the circulation, low physical stability in body fluids and at infection sites due to the degradation by enzymes and stomach acids, limited bioavailability through binding with (plasma) proteins and other molecules, and low tissue penetration ,16,17. OIn this review, we will discuss the main chemical characteristics, the release profile and, most importantly, the antibacterial and antibiofilm activities as well as toxicity of the lipid and polymeric AMP delivery systems and coatings reported in the literature. In addition, we attempt to identify which delivery system and/or coating is profitable for AMP delivery in the fight against and/or prevention of the major hard-to-treat infections, including bloodstream and deep-seated infections, catheter-related and implant-associated infections, pulmonary tract infections, and complex wound infections (such as diabetic ulcers and burn wounds).This review is based on studies in English without restrictions regarding year of publication; however, we mainly focused on the recent literature spanning from 2016 to 2021. References were sourced from electronic databases PubMed, Embase, Web of Science and Cochrane Library. A two-stranded search strategy was applied, where the main search terms included “antimicrobial peptides”, “type of nano formulations”, “drug delivery system”, “coatings” and “infection”. Subsequently, titles and abstracts were screened to obtain the relevant literature.Drug delivery technology has advanced significantly since the introduction of the first controlled-release formulation in the 1950’s , and is Provided below is an overview of lipid and polymeric nanoparticle delivery systems encapsulating AMPs and coatings developed in the last 5 years together with a discussion regarding some of the advantages and limitations of these systems against in vitro and in vivo infections.Liposomes are vesicle bilayers composed of natural phospholipids and cholesterol with an aqueous core . HydrophPseudomonas aeruginosa compared to a colistin solution in vitro. In vivo studies of P. aeruginosa tracheal-infection-bearing mice showed that treatment with colistin-loaded liposomes resulted in the survival of 50% of the mice up to 96 h post-infection, while none of the mice treated with empty liposomes or the colistin solution survived longer than 24 h post-infection. In addition, colistin-loaded liposomes reduced systemic exposure of the drug in mice, improving the safety of the AMP .,76.75,76ffective . OverallMicelles are self-assembled spherical structures composed of single layer lipids that are organized with their hydrophilic head pointing towards the water phase and their hydrophobic tails accumulating in the center of the micelles . AlthougAcinetobacter baumannii and P. aeruginosa and biofilms of the latter, while reducing cytotoxicity to two types of epithelial cells and reducing the hemolytic activity of polymyxin B [S. aureus biofilm, while cytotoxicity was reduced against red blood cells and human peripheral blood mononuclear cells compared to a peptide 73c solution. Surprisingly, the encapsulation of peptide 73c into these micelles resulted in enhanced activity in an in vivo abscess mice model infected with MRSA, compared to other aurein-derived AMP-loaded micelles [P. aeruginosa infection in zebrafish and MRSA infection in mice. In this murine abdominal infection model, DP7-C also stimulated defensive immune reactions [The few AMP-loaded micelles described in the literature are summarized in ymyxin B ,83. Moremicelles . Similareactions . Overallco-glycolide) (PLGA), are a subtype of synthetic biopolymers characterized by hydrophilic and hydrophobic blocks of chemically different polymer units linked together by covalent bonds that undergo phase separation, which results in the formation of polymeric nanoparticles (NPs) [Synthetic biopolymers are commonly utilized for the fabrication of polymeric nanoparticles to improve the efficacy and reduce the toxicity of AMPs. Polyesters, e.g., poly(lactic acid) (PLA) and poly(lactide-es (NPs) . Polyestes (NPs) . Additioes (NPs) . Polysaces (NPs) . MoreoveP. aeruginosa lung infection by up to ~17-fold compared to the peptide solution [E. coli [50) of MRSA for G17NPs and G19NPs, respectively; and the MIC50 of E. coli showed a 2-fold decrease for both formulations 8 h post-treatment [E. coli with PLGA-encapsulated HHC10 in a concentration-dependent manner and higher cellular uptake in mouse macrophages compared with a peptide solution 12 h post-treatment [E. coli-infected cells in vitro. HHC10 showed less cytotoxic effects on mouse macrophages compared to a peptide solution at equivalent amounts 24 h post-treatment. These findings suggest that incorporation of HHC10 in PLGA could reduce the probability of inducing the membrane lysis of mammalian cells due to the higher intracellular uptake observed compared with a peptide solution. Sharma et al. functionalized PLGA NPs, encapsulating IDR-1018 with a biofilm penetrating ligand, N-acetyl cysteine (NAC), to improve their antibiofilm activity against Mycobacterium tuberculosis [M. tuberculosis-infected macrophages compared to nonfunctionalized PLGA NPs. NAC-coated PLGA NPs displayed a significantly higher bacterial killing activity than noncoated PLGA NPs, which is attributed to higher uptake of NAC-PLGA NPs combined with additive antibacterial activity of the NAC ligand.PLGA is of particular interest for AMP delivery and tissue regeneration purposes as lactate, a breakdown product of PLGA, is known to accelerate neovascularization and enhance wound healing . Additiosolution . Cheredd[E. coli . Additio[E. coli . The pepreatment . Additioreatment . The bacrculosis . The res50 with peptide-loaded PLA/PLGA NPs (MIC50 = 0.5 µM) compared with the peptide solution (MIC50 = 10 µM) against E. coli, S. aureus and P. aeruginosa 8 h post-treatment. Moreover, there were no significant differences in antibacterial activity between the two types of polymeric nanoparticles, apart from killing against E. coli where PLA antimicrobial activity was higher (MIC50 < 0.5 µM) compared to PLGA nanoparticles (MIC50 = 1–10 µM). The higher antimicrobial activity observed with encapsulated peptide could be mediated by electrostatic interactions between the positively charged PLA/PLGA nanoparticles (20–30 mV) and negatively charged surface of Gram-negative and Gram-positive bacteria, which may lead to a higher local peptide concentration on the surface of bacteria [PLA is another important synthetic polymer commonly used for biomedical application due to its biocompatibility, tailorable properties and established fabrication techniques. The chemical structure of PLA consists of monomer units connected by ester bonds, which are naturally degraded by hydrolysis . Cruz etbacteria ,98.l-lactic acid-co-d,l-mandelic) acid nanoparticles (poly(LA-co-MA) NPs) are a class of biodegradable polymers in which the polymer backbone is composed of repeating units connected by anhydride bonds. The degradation of polyanhydrides consists of two stages with an initial swelling at the surface of the matrix without degradation (induction period), followed by an erosion of oligomers by swelling of the main chain [co-MA) NPs with a diameter of around 275 nm and an encapsulation efficiency and loading capacity of 92% and 8%, respectively. They showed poly(LA-co-MA)-encapsulated cathelicidin-BF-30 had an antibacterial activity profile similar to a peptide solution on several bacterial species, including E. coli and S. aureus, using the in vitro zone inhibition test [co-MA) NPs could be a potential drug carrier for AMPs.Although AMP-loaded PLGA and PLA DDSs show both structural stability and antimicrobial activity, the initial burst release is undesirable for clinical application due to possible side effects, which may reduce their clinical usability. Additionally, the sustained release phase that follows the burst phase might not achieve the MIC needed to kill bacteria. Polyanhydrides, such as poly peptide formulated into PLGA/chitosan composite microspheres on oral bacteria (F. nucleatum) for up to 80 days post-treatment using the inhibition zone assay [Chitosan is a natural biodegradable and biocompatible polymer that can provide controlled release, improved bioavailability and safety of AMPs. Chitosan nanoparticles have been proven to effectively enhance cellular penetration, intracellular retention and subcellular distribution of AMPs . Moreovene assay . ChitosaArthrobacter sp. and Bacillus subtilis) and two Gram-negative bacteria (E. coli and Klebsiella spp.) using the agar-diffusion assay [Pectin is a naturally occurring polysaccharide consisting of mainly D-galacturonic acid units joined by glycosidic linkage units . Pectin on assay . They shA major drawback of nontargeted polymeric nanoparticles is the lack of chemical functionalities on the surface to induce site-specific interactions, such as adhesion, internalization and penetration through biological membranes. Additionally, delivery of hydrophilic molecules  is limited by poor encapsulation and a large burst release of encapsulated drug within the first few hours. The initial burst release is mainly due to desorption of hydrophilic AMPs from the surface of the biomaterial devices composed of hydrophobic polymers , due to The functionalization of polyester-based therapeutics is of particular interest because it allows the fine tuning of particle characteristics, such as hydrophilicity, surface charge and release at the site of infection. This can enhance targeted drug delivery of AMPs, for example by promoting intracellular drug delivery of AMPs and also allowing accumulation at infection sites. Furthermore, an additive or even synergistic antimicrobial effect can be achieved by encapsulating the AMP in a polymer with intrinsic antimicrobial properties, such as chitosan. Functional groups can be incorporated on the aliphatic backbone of the polyesters via direct conjugation or with additives during the formulation process to elicit specific drug or cell interactions. Polymeric drug delivery systems have unique properties and functions which make them suitable for AMP delivery, though certain issues related to stability, toxicity and loading/encapsulation need to be addressed by modifying the formulation process for effective AMP delivery. To date, there are no marketed polymeric nanoparticle formulations for AMPs.2– and –SO3H on the polymer backbone [In recent years, nanogels have received increased attention as a versatile delivery system due to their unique potential resulting from the combined features of hydrogels and nanoparticles. These soft nanoparticles are three-dimensional cross-linked polymeric networks made of water-soluble natural or synthetic polymers that have the ability to absorb high amounts of water or biological fluids into the formed network while maintaining their structure  111,112112. The backbone , which pbackbone ; nanogelbackbone . The prebackbone ,116. Thebackbone , which, The low interfacial tension and the deformability of nanogels can potentially minimize nonspecific protein adsorption , and impco-methacrylic acid) and poly(N-isopropylacrylamide) (PNIPAAm)  . Both caSalmonella infections in vivo [Chitosan-based nanoparticles do not self-assemble, instead they form noncovalent interactions with anionic molecules to form polyelectrolyte complexes . This ge in vivo . A histoM. tuberculosis. Sharma et al. showed that encapsulation of the peptide pep-H in chitosan nanogels resulted in the formation of nanoparticles with a cationic surface charge, which then allowed an 80% reduction of intracellular bacterial load in comparison to equivalent concentrations of peptide in solution, which only showed a 12% reduction in intracellular M. tuberculosis load [Utilizing chitosan as the nanogel matrix often provides a positive surface charge, which may increase macrophage uptake, making it a suitable delivery system for treatment of intracellular infections, such as sis load . Chitosasis load . Howeversis load . For sucl-lysine cross-linked HA nanogels to illustrate rapid internalization of this system in alveolar basal epithelial cells followed by endosomal escape to allow delivery of the drug intracellularly [M. tuberculosis infections in macrophages [P. aeruginosa and S. aureus intracellular infections [P. aeruginosa [Two most commonly used natural anionic polymers for the formulation of AMPs are hyaluronic acid and alginate . Both pollularly . Silva erophages . The nanfections . Additiofections , well knfections . HA-baseruginosa .2+, Sr2+ or Ba2+ in aqueous solutions to form soft nanoparticles, whereas monovalent cations, as well as Mg2+, do not induce cross-linking [Another natural anionic polymer suitable for the delivery of AMPs is alginate, a naturally occurring polymer derived from brown algae, which is increasingly used in both food and pharmaceutical applications. Alginate can be ionically cross-linked by addition of divalent cations, such as Ca-linking . A range-linking . The ant-linking . This isco-methacrylic acid) have been described. Masuda et al. encapsulated the peptide E5 by forming a polyelectrolyte complex with (PAA-g-PNIPAAm) and found that using the thermoresponsive PNIPAAm side chain allowed a better control of the membrane-disrupting activity of the delivery system on a bacterial membrane model [co-methacrylic acid) protected the encapsulated peptides LL-37 and DPK-060 from degradation by infection-related proteases and in the case of peptide DPK-060, improved the antimicrobial activity of the peptide as determined by MIC [A vast range of synthetic polymers for the preparation of drug delivery systems has been developed, though few reports are available regarding the encapsulation of AMPs in such systems. Among synthetic polymers used for this purpose, poly-poly(N-isopropylacrylamide) (PAA-g-PNIPAAm) and poly  and 33 mg of cefazolin combined with 4 mg of gentamicin administered subconjunctively once a day. Others adapted the polydopamine coating technique by using a gelatin-based hydrogel modified with DOPA motifs, further referred to as Gel-DOPA, and functionalized it with HHC-36 [S. aureus, S. epidermidis, P. aeruginosa and E. coli were reduced by >99%. In addition, the peptide released from Gel-DOPA-coated titanium plates was highly effective in killing these bacteria.The antibacterial effects of the AMPs immobilized in thin layer polymeric coatings are summarized in ruginosa . In agreruginosa . Resultsh HHC-36 . When apco-APMA) copolymer brushes were optimal for AMP immobilization [co-APMA) copolymer brushes attached to polystyrene nanoparticles [P. aeruginosa and S. aureus; however, the coated peptides were less effective than peptides in solution. In addition, S. aureus adherence onto a polymer brush enriched with E6 and coupled to titanium was moderately (10–40%) reduced compared to uncoated titanium. Others developed polymeric brushes composed of AMP-functionalized block copolymer Pluronic F-127 immobilized onto silicone rubber surface by dip-coating [S. aureus, S. epidermidis and P. aeruginosa and killed those bacteria that adhered to the surface. Yu et al. used an antiadhesive brush poly(DMA-co-APMA) copolymer coating on polyurethane to immobilize peptide E6 [P. aeruginosa and S. aureus and Staphylococcus saprophyticus and reduced planktonic bacterial growth by up to 70%. Importantly, in a mouse urinary catheter infection model the AMP-coated catheter was highly effective in preventing P. aeruginosa infections by reducing bacterial adhesion to the catheter in urine by 4 logs. Mishra et al. immobilized a potent AMP onto a silicone catheter using an allyl glycidyl ether brush and polyethylene glycol-based chemical coupling [2 and the coated catheter exhibited excellent antimicrobial activity against E. coli and Enterococcus faecalis and prevented biofilm formation by these bacterial species. These antibacterial properties were maintained over a period of at least 4 days. Furthermore, Monteiro et al. conjugated the peptide Chain201D and a control peptide to tetra(ethylene) glycol-terminated self-assembled monolayers (EG4-SAM) activated by carbonylimidazole on gold surfaces [S. aureus and E. coli. For example, Godoy-Gallardo et al. immobilized hLf1-11 on titanium surfaces by silanization and with polymer brushes prepared by surface-initiated atom transfer radical polymerization [Streptococcus gordonii and Porphymonas gingivalis viability in the adherent population significantly. Based on their antiadhesive, antibacterial and biocompatible properties polymeric brush-AMPs coatings are interesting candidates for further development as coatings for metal implants and catheters.Polymeric brushes are macromolecular structures with polymeric chains that are from one end chemically coupled to surfaces and from the other end coupled to AMPs. The polymeric brush provides flexibility between the AMP and the surface and increases the density of the AMP on the surface, thereby reducing the impact of surface confinement. A number of studies have utilized the polymeric brush technology to equip titanium surfaces and silicone surfaces with an AMP . Gao et lization . Yu et aarticles . These A-coating . These aptide E6 . This sucoupling . The amosurfaces . Chain20rization . Resultsrization . They re2 and release the peptide over a period of 10 days. Importantly, this peptide coating prevented S. aureus from adhering to the surface. Raman et al. produced a multilayered coating consisting of poly-L-glutamic acid and poly-L-lysine incubated with AMP onto the luminal surface of catheters [Candida albicans and to decrease C. albicans biofilm formation by 83% in vitro. Kazimzadeh-Narbat et al. prepared a three-layered coating on titanium surfaces consisting of TiO2 nanotubes oriented vertically on the titanium surface using the drop and dry technique, a thin layer of calcium phosphate by electrolytic deposition, and finally a film of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine, with each layer being impregnated with the HHC-36 peptide [S. aureus and P. aeruginosa in the disk diffusion assay. In vitro, this peptide was released from the coating in 5 days after an initial burst release of 27%. In another study, Tet213 was linked to collagen IV and titanium surfaces were coated with multilayers of the AMP-functionalized collagen using a layer-by-layer technique [S. aureus and P. gingivalis during the entire interval by 40–55%. Importantly, this coating inhibited S. aureus biofilm formation considerably. Another highly effective multilayered coating system comprised three layers of PLGA, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine and cholesterol (PLEX) mixed with 10% OP-145 peptide [S. aureus-inoculated silicone elastomer implants in mice. In a follow up study using a murine model for biofilm-associated infection, Riool et al. showed that other, closely related, antimicrobial and antibiofilm peptides (SAAPs) incorporated in a five-layered PLEX coating on titanium/aluminum/nobium (TAN) implants significantly reduced the number of culture positive implants and resulted in ≥3.5 and ≥1.5 log lower S. aureus counts on the implants and peri-implant tissues [S. aureus in this model; both peptides reduced implant colonization by 2 logs, whereas the SAAP-276 PLEX coating, but not the SAAP-145 PLEX coating, decreased tissue colonization by 1 log. Together, these data indicate that implant colonization by MRSA can be prevented by PLEX coatings releasing SAAPs. It is tempting to state that SAAPs applied in PLEX coatings may be more effective than when administered as peptide solution.The layer-by-layer coating strategy creates multilayer AMP reservoirs on surfaces. This technique is very powerful in protecting metal surfaces and catheters from colonization by biofilm-forming bacteria. Moreover, the multilayer composition permits a controlled release of the AMP over a long time span. Several studies have successfully applied this layer-by-layer technique to develop a coating that protects surfaces from bacterial colonization by biofilm-forming bacteria . For exaatheters . The AMP peptide . The coaechnique . The pep peptide . They re tissues . This PLFinally, polymeric AMP coatings provide many advantages over adsorption or direct immobilization of AMP onto metal and organic  materials. It should be noted that the efficacy of coatings equipped with AMPs in vitro are often not challenged in biologically relevant conditions, such as urine, in the presence of proteins, cells of the immune system or under dynamic/flow conditions etc. However, several coatings have been tested in animal models .This review aimed to provide an overview of lipid and polymeric AMP delivery systems and coatings developed in the last 5 years and to discuss some of the advantages and limitations of these systems against in vitro and in vivo infections. In the above content, we have critically evaluated the antimicrobial properties of polymeric and lipid-based delivery systems and coatings for AMPs in efficacy studies in vitro as well as in vivo. Here, a perspective for developing such formulations into therapeutics will be provided by discussing important limitations of AMP-based nanoformulations and providing several suggestions and recommendations, which may expedite AMP-based DDSs research to the clinic.Despite AMP-based DDSs displaying several advantages compared to their nonformulated counterparts, significant challenges remain to be addressed to produce a viable treatment product for clinical applications against infectious diseases. One of the limitations is a lack of standardized testing in biorelevant conditions, which would allow an effective evaluation and comparison between DDSs. Another important aspect that needs to be addressed for development of these systems into therapeutics is the preparation and evaluation of shelf-stable macroformulations. Both challenges are discussed in more detail below.Although there are several publications on the preparation and in vitro characterization of DDSs, research is lacking on fully testing the activity of AMP-loaded formulations. One of the most commonly occurring limitation is the improper use of controls—in many cases the peptide-loaded systems are compared only to nonloaded systems or no treatment and not compared to the peptide solution. Such comparisons will often show improved antimicrobial efficacy, but do not indicate if peptide activity was lost upon encapsulation.To determine the antimicrobial activity and toxicity of the delivery system in a manner that allows comparison between studies, standardized in vitro testing in combination with in vivo evaluation is highly necessary. Currently, antimicrobial testing is mainly performed by broth microdilution MIC testing or zone inhibition tests. These are simple and well-established methods; however, both do not necessarily indicate the killing of bacteria, but rather determine the inhibition of bacterial growth. In addition, the MIC is obtained after a fixed overnight incubation time  and is unable to distinguish between partial or complete inhibition and between bactericidal or bacteriostatic mode of action . Most imMany complex in vitro setups have already been developed, such as killing assays on planktonic bacteria as well as immature and mature biofilms. These assays could be further expanded using a relevant microenvironment that mimics the clinical situation to understand the suitability of a delivery system for a given application. This could include incorporation of host cells, for example in 3D collagen-elastin matrix models, immune cells and factors, as well as biological fluids, for example proteolytic enzymes, urine or plasma proteins, as the presence of these components are known to affect the antimicrobial activity of AMPs in vivo ,173,174.Moreover, once sufficient evaluation in vitro and ex vivo has been performed, in vivo experiments are needed to evaluate not only antimicrobial efficacy and safety, but also the pharmacokinetics and pharmacodynamics of the DDS, as well as immunological responses. The choice of animal model should be carefully considered before selection. For topical application, this may be rather simple as a range of wound models on the skin of mouse and pig have been developed . HoweverAlthough both nanoparticle-based systems and coatings have shown impressive activity in vitro and some in vivo, the development of a shelf-stable liquid, gel or solid dosage form is still necessary for the translation of a DDS to the clinic. The shelf stability of AMP-based products is important not only due to the sensitive nature of AMPs, which are prone to degradation and hydrolysis, but also due to interactions that may occur between the AMP and the macroformulation. For example, the incorporation of SAAP-148 in a hypromellose gel carrier has shown to reduce the peptide’s performance, with increasing viscosity resulting in a reduced activity . Dijkstel-lysine with catechol showed significantly reduced bacterial burden by more than 4 logs in multidrug-resistant A. baumannii-infected burn wounds [Other macroformulations have shown more promising results. Hydrogels prepared by cross-linking the AMP epsilon-poly-n wounds . Chitosan wounds ,185. Addn wounds , which iAnother important question relates to the choice of AMP-based DDSs for the treatment of different infections. In this section we attempt to identify which lipid or polymeric AMP delivery system or coating is profitable in the fight against and/or prevention of the major hard-to-treat infections. For treatment of infections that are caused by bloodstream-circulating bacteria and deep tissue infections, a DDS that protects the peptide from enzymatic degradation and rapid removal from the circulation and that can be administered systemically is most desirable. Soft nanoparticles, such as nanogels, may be advantageous due to their deformability, which may offer enhanced circulation and aid transport of the delivery system through tissues and to the infection site . FusogenAll medical interventions, in particular catheter applications or implantations, are associated with the risk of introducing possible infections. Therefore, there is a need for efficient infection prevention, putting coatings that release AMPs to prevent or treat biomaterial-associated infections in high demand. Coating the implanted material with AMPs may prevent infection occurrence, minimizing post-surgical complications, and the released peptide can eliminate the bacteria already residing in tissues surrounding the implant. Although impressive results have been observed for a range of coatings in vitro, layer-by-layer coatings have shown to be particularly effective for long-term infection prevention, with sustained release reported for up to 4 months for some layer-by-layer coatings .M. tuberculosis, a delivery system that can be internalized in the epithelial cells is desirable. Smaller nanoparticles (>20 nm) induce uptake without requiring endocytic mechanisms [In the case of specific lung infections, such as chronic bronchitis or cystic fibrosis, a topical administration route by inhalation could be desirable to achieve high local AMP concentrations. Upon inhalation, the AMP has to permeate through the lung mucus and bacterial biofilm to reach the bacteria. In these cases, a mucus-penetrating nanoparticle system could be advantageous, due to the continuous removal of lung mucus by mucociliary clearance. Such a system would be able to diffuse through mucus and deliver high concentrations of AMP in close proximity of the bacterial infection. Additionally, if the aim is treatment of an intracellular infection, such as chanisms , though chanisms . As a rechanisms . Similarchanisms ,195. Thechanisms ,197. IntComplex wound infections, such as burn wound infections, fracture-related infections and prosthetic joint infections, are associated with biofilms, which protect bacteria from host immune defenses and significantly increase antibiotic resistance. Often, it is unavoidable to treat these wounds by surgical removal of most harmed tissue followed by aggressive antibiotic treatment. For such infections, a classic treatment with cationic antibiotics, such as gentamicin or AMPs, has shown little success due to matrix binding . A DDS tAs outlined above, the suitability of a DDS for a given purpose is currently based on reports primarily regarding a single delivery system. Contrasting reports on required properties for some applications only emphasize the need for comparative studies between various DDSs to determine the most desirable properties for this purpose. Back-to-back comparisons of DDSs performed in standardized conditions and on the same models are needed. Such information enables selection of the most suitable DDS for a given application and will substantially increase chances of developing a formulation that can be used in the clinic.Despite significant progress in the area of AMP drug delivery technology, further work is needed for nanoparticle-based systems or coatings to be developed into clinical therapies. One significant limiting property of many currently described DDSs is a relatively low encapsulation efficiency. This is associated with additional workload due to purification steps that need to be included and a high cost due to significant peptide loss during purification. The encapsulation efficiency can be improved by varying formulation parameters to suit the drug for encapsulation, as has been shown for liposomes , PLGA naMost recently, the trend in the design of AMP delivery systems seems to focus on the design and development of hybrid AMP delivery systems, where the particle surface is functionalized with PEG, biofilm-penetrating ligands, or cell-penetrating peptides to obtain improved targeting and intracellular uptake and simultaneously overcome any possible disadvantages associated with the system itself. Improving existing delivery systems in a precise and targeted way is an excellent approach to improve the targeting of AMPs to their site of action, while building on existing knowledge of currently available delivery systems. Finally, standardized testing and shelf-stable forms of the developed systems are lacking. Therefore, there is an urgent need for clear guidelines on in vitro and ex vivo testing, as well as a need for more relevant in vivo infection models, preferably testing shelf-stable forms of the products, to assess the safety issues and performance of these formulations.The increasing development of bacterial resistance to traditional antibiotics has directed research attention to alternative therapies, such as AMPs. However, nonoptimal physicochemical properties, insufficient efficacy and toxicity data, as well as costs, have limited the translation of a large part of AMPs into therapeutic products. Nanoparticle-based AMP therapies and coatings have shown promising results in vitro, aiding delivery of AMPs, not only through bacterial biofilms and in close proximity to bacteria, but also into cells for the treatment of intracellular infections.The progress in the fields of drug delivery and nanotechnology has led to a vast array of novel nanoparticle systems and coatings, which may allow efficient delivery across biological membranes and an improvement of the antibacterial activity at the site of infection. Nonetheless, the majority of available reports are in vitro-based findings and many challenges still need to be addressed, such as the lack of reproducible infection-specific in vitro and in vivo models, as well as specific guidelines and standards for testing the safety, efficacy and performance of nanoparticle-based therapeutics. Additionally, the translation of these early-stage drug development findings to shelf-stable solid dosage forms is crucial. Overcoming these obstacles will lead to safer and more efficient nanoparticle-mediated AMP therapies entering the clinical phases of drug development.


There is a pressing need for effective therapeutics for coronavirus disease 2019 (COVID-19), the respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. The process of drug development is a costly and meticulously paced process, where progress is often hindered by the failure of initially promising leads. To aid this challenge, in vitro human microphysiological systems need to be refined and adapted for mechanistic studies and drug screening, thereby saving valuable time and resources during a pandemic crisis. The SARS-CoV-2 virus attacks the lung, an organ where the unique three-dimensional (3D) structure of its functional units is critical for proper respiratory function. The in vitro lung models essentially recapitulate the distinct tissue structure and the dynamic mechanical and biological interactions between different cell types. Current model systems include Transwell, organoid and organ-on-a-chip or microphysiological systems (MPSs). We review models that have direct relevance toward modeling the pathology of COVID-19, including the processes of inflammation, edema, coagulation, as well as lung immune function. We also consider the practical issues that may influence the design and fabrication of MPS. The role of lung MPS is addressed in the context of multi-organ models, and it is discussed how high-throughput screening and artificial intelligence can be integrated with lung MPS to accelerate drug development for COVID-19 and other infectious diseases. Through modeling specific aspects of the disease state, MPS can play a key role in accelerating, de-risking, or supplementing the development of novel treatments for various pathologies. Excellent reviews have been published on general organ-on-a-chip and lung-on-a-chip models for drug development . Us. Us59]. Zinc and zinc metalloenzymes are further alternative targets for drug development against SARS-CoV-2 entry. Facilitating zinc entry is a proposed mechanism for chloroquine and hydroxychloroquine, which are controversial antimalarial drugs repurposed for COVID-19 treatment as part of the SOLIDARITY trial. Acting as an ionophore, they increase intracellular lysosomal zinc concentrations, which has been shown to inhibit viral RNA-dependent RNA polymerase , 64. Thec) is being explored [The inflammatory component of COVID-19 is a complex response to infection by SARS-CoV-2 in vascular connective tissue. A sequence of cell migration into the extracellular matrix occurs due to viral infection—this sequence begins with neutrophils followed by lymphocytes and macrophages , 75. Infexplored , 83–85. explored , 87. Theexplored , 83, 88.explored , 89. Anoexplored , 82, 89.explored . Inflammexplored –93. IL-1explored , 85. In explored . In ordeThe increased production of proinflammatory cytokines activating the coagulation reaction cascade leads to coagulopathy in COVID-19 patients. The coagulopathy seen in COVID-19 has the characteristics of sepsis-induced coagulation (SIC), as well as of severe disseminated intravascular coagulation (DIC) , 96. The+ absorption drives the removal of liquid from the alveolar lumen [–) follow the actively absorbed Na+, resulting in a local osmotic gradient across the epithelium and water flowing along this gradient, which occurs primarily though alveolar type I cells that have the highest osmotic permeability of any cell type [At equilibrium, the active removal of liquid from the alveoli equals the inflow due to Starling forces, and the depth of liquid in the alveolar lumen is constant at approximately 0.1 µm . Amiloriar lumen . Anions ell type . It is nell type , 107. Ifell type . Lung-onell type . The TRPell type .Viral-induced pulmonary fibrosis contributes to both an immediate reduction in lung function and an increased risk of developing pulmonary fibrosis . EpithelA fibrotic phenotype is typically achieved by the addition of a lung fibroblast population and a fibrosis activation process (TGF-β supplementation) within type I collagen-based matrices and can be quantified in different ways depending on the system. In one approach, human small airway epithelial cells (SAECs) were combined with endothelial cells and normal lung fibroblasts within a multi-layer perfusable PDMS device with a distinct design that facilitates cell–cell interactions . A fibroThe cooperative responses of innate and adaptive immune cells within the lungs provide antibacterial, antifungal and antiviral immunity and are a source of significant paracrine signaling. Local macrophages and lymphocytes make up 90% and 10% of effector immune cells, respectively, within the lung parenchymal tissue . AlveolaRecent efforts have aimed at recreating specific immune–epithelial cell interactions within lung-on-a-chip systems to better model intrinsic behaviors. In one specific study, primary bronchial or small airway epithelial cells were seeded on collagen-coated Transwell membranes and cultured under perfusion with an air–liquid interface to create matured upper or lower respiratory tract models, respectively . AntigenBronchus-associated lymphoid tissues are found within the bronchioles and act as the local sites of B and T cell accumulation, antigen presentation and activation and consequent differentiation . This prIn order to best evaluate therapeutics against COVID-19 and its associated tissue trauma, the development of lung models must combine design criteria spanning relevance to (1) native tissue structure and function and (2) disease-specific or multi-disease pathology. Based on our review of relevant scientific literature, we believe that the modular platform of lung MPS is the most promising candidate, owing to the capacity of incorporating multiple cells types, biochemical and biophysical stimulation, and diverse characterization and output parameters in 3D microenvironments . Based oAs previously discussed, novel models that integrate SARS-CoV-2 viral entry, immune responses and disease-specific pathology will be the best option to evaluate therapeutics across early- to late-stage infections. The first step is to employ lung primary cells  or cell lines that balance physiological relevance with ACE2 and TMSPR2 expression. Functions of inflammation and immunity can be introduced via exogenous cytokines , macrophages, neutrophils and/or lymphocytes. A key design component, via either membrane pores or compartmentalization, is the capacity for immune cell migration and maturation to enact direct and paracrine effects on lung tissue cells. We earlier discussed the most prevalent complications associated with COVID-19, namely coagulation, pulmonary edema and fibrosis. A corresponding disease-specific phenotype can be introduced and modeled via biochemical stimulation , additional cell types, genetic modifications, or changes in material stiffness. The direct integration or circuit connection of multiple disease elements should allow for therapeutic intervention and quantifiable disease outcomes for treatment evaluation in a higher-throughput fashion.The integration of these intricate systems within a single tissue model represents a formidable challenge. However, COVID-19, which is currently affecting a rather large portion of the population with relatively unexplored long-term side-effects, justifies these efforts. An ideal system that can accurately determine effective therapeutics for each disease stage is therefore expected to have a significant impact.While lung microphysiological systems play an important role in testing leads for COVID development, interfacing a lung MPS device with multi-organ MPS or body-on-a-chip is necessary for determining secondary and systemic effects . SystemiThe process of creating an in vitro model of COVID-19 infection presents unique challenges. First, the cellular components need careful selection. For instance, the SARS-CoV-2 infection induces cellular damage and an aberrant immune response leading to microenvironmental changes seen in the vascular compartment of the MPS, including elevated glucose, raised creatinine and markedly increased overall protein levels due to cell debris . AlthougOn the other hand, the biophysical properties of MPS should be tailored to mimic the in vivo tissue environment. For instance, there are significant differences in the elasticity of the components of lung MPS models, including the PDMS sidewalls, the material supporting the basement membrane of the alveolar epithelial and endothelial layers, as well as the cells themselves and their deposited ECM. Therefore, it should be considered that the elasticity changes with the disease state in some of these components. Lung elasticity is determined by the elastin content, patterning and the level of collagen isotypes –148. Theqe (mg/g) represents the amount of compound (as solute) adsorbed, Qm (mg sorbate/ g sorbent) represents the maximum adsorption capacity, Ka (L/mg) represents the affinity constant for adsorption, Ce represents the concentration at equilibrium (mg/L) and n represents the index of heterogeneity of the surface. The relation is semiempirical, allowing the aggregation of complex effects in a functional MPS. Alternative materials that exhibit low drug binding, such as polysulfones, polycarbonates and thermoplastics, can also be used in place of PDMS [The surface properties of MPS materials also need to be considered. Polydimethylsiloxane has many desirable properties, such as transparency, gas permeability, flexibility, ease of device manufacture, as well as biodegradability . However of PDMS , 161. Bi of PDMS –164. Tre of PDMS , 161. Lo of PDMS . Alterna of PDMS .The conceptual basis of microfluidic flow, i.e., when small volumes are driven through bifurcating micron-scale channels, is readily applied to high-throughput screening. In early cancer modeling chip systems, 100 wells of HeLa cells arranged in a 10 × 10 array were subjected to a microfluidically generated concentration gradient of analyte for dosage–response analysis . RecentlThe role of artificial intelligence (AI) in accelerating drug development and repurposing is an intriguing field that has attracted much interest. In other COVID-19-related areas, artificial intelligence and machine learning have proven useful for applications such as molecular design, data mining and image analysis . ConvoluThe application of MPS to understand lung physiology and disease states is expanding at a rapid pace. Their role alongside other co-culture systems, such as organoids and Transwell systems, in drug discovery that more closely follows in vivo physiology and pathology, should become the next rising trend. The efficient adaptation of these in vitro models for the development and characterization of COVID-19 treatments will likely benefit patients on an unparalleled scale and lay the groundwork toward preparedness for the future, where the increasing global connectedness could lead to pandemics of similar or even greater magnitude than that of the current coronavirus outbreak.


The fundamentals of how protein–protein/RNA/DNA interactions influence the structures and functions of the workhorses from the cells have been well documented in the 20th century. A diverse set of methods exist to determine such interactions between different components, particularly, the mass spectrometry (MS) methods, with its advanced instrumentation, has become a significant approach to analyze a diverse range of biomolecules, as well as bring insights to their biomolecular processes. This review highlights the principal role of chemistry in MS-based structural proteomics approaches, with a particular focus on the chemical cross-linking of protein–protein/DNA/RNA complexes. In addition, we discuss different methods to prepare the cross-linked samples for MS analysis and tools to identify cross-linked peptides. Cross-linking mass spectrometry (CLMS) holds promise to identify interaction sites in larger and more complex biological systems. The typical CLMS workflow allows for the measurement of the proximity in three-dimensional space of amino acids, identifying proteins in direct contact with DNA or RNA, and it provides information on the folds of proteins as well as their topology in the complexes. Principal CLMS applications, its notable successes, as well as common pipelines that bridge proteomics, molecular biology, structural systems biology, and interactomics are outlined. Decades of research into the cell biology, molecular biology, biochemistry, structural biology, and biophysics have produced a detailed understanding of individual DNA/RNA/protein molecules, and their interconnected networks. A great diversity of techniques has emerged for studying their structural interactions. However, even more complex, the structures of these workhorses from the cells are themselves dynamic, converting from one dominant form to another based on the proportions of particular proteoforms present for any given biomolecules. Accordingly, beyond the protein–protein/DNA/RNA interaction landscape, there is an entire universe to explore with respect to their structure and dynamics. One such high-throughput technique has emerged as a dominant player in understanding both interaction landscapes and their resulting protein/DNA/RNA structures, namely cross-linking mass spectrometry (CLMS). This review covers different methods that are available to study protein–protein/DNA/RNA interactions, and provides vital insight into CLMS, a collection of methods that are perfectly suited to achieve a better understanding of intra- or inter- molecular interactions.Protein–protein interactions (PPIs) play a crucial role in all biological/biomolecular processes to understand the molecular mechanism of relevant protein molecules; hence, they are often termed the workhorses of cells. Over 80% of proteins do not function in isolation, but rather exist in interactions with one another to obtain stable or transitory complexes, as demanded by their observed function ,2. PPIs Mass spectrometry (MS) is one of the powerful approaches available that is useful in several areas beyond CLMS , and itFundamentally, the mass spectrometry methods simply measure a mass-to-charge ratio of an ion. Initially, analytes are ionized and then transferred into the gas phase prior to their separation according to mass-to-charge ratios in a mass analyzer. Subsequently, ions that emerge from the mass analyzer are recorded using a detector. The most common way to ionize a peptide or protein sample is electrospray ionization (ESI) . HoweverQuantitative cross-linking mass spectrometry (QCLMS) approaches investigate protein structures as well as the dynamics of their interactions ,49,50,51Isobaric Tags for Relative and Absolute Quantitation/Tandem Mass Tag (iTRAQ/TMT) are chemical labeling, which are introduced to peptides after protease digestion, which allow relative protein quantitation via MS2 or MS3 encoded data. Nowadays, TMT/iTRAQ labeling enables comparison of up to 16 samples  in a single MS analysis. Notably, the iTRAQ/TMT reporter ions used for quantitation are cleaved from labeled peptides during fragmentation by way of collision-induced dissociation allowing for the quantitation from fragment ion relative intensities. Yu et al. , implemeMoreover, techniques, such as proximity-dependent biotin labeling (or BioID technique) in living cells, help to understand the plasticity of protein networks within heterogeneous cellular populations. In combination with nanopore technology, such an approach could help tackle pending biological questions, e.g., the identification of peptidyl-prolyl isomerases (PPIases) substrates. PPIases substrates preserve their primary structure/molecular mass, as well as the cis and trans isomers of the proline peptide bonds of the substrates interconversion by PPIases being the sole change. This subtle modification triggers important changes to the substrate’s fate, such as subcellular translocation, degradation, or rewiring of their protein–protein interaction networks. PPIase enzymes act as central molecular switches, as exemplified by the peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) that has been extensively studied and showing its involvement in multiple diseases ,59,60. HE. coli) as the host system, functionally important post-translational modifications and interacting protein partner associations may be lost during this process. Thus, various biochemical approaches must be explored when beginning a new project in order to directly enable the isolation of endogenous protein complexes from cells or tissues .,79.78,79organism ,78,79. MThree main chemical approaches that involve MS analysis on the peptide level are: (i) the exchange of labile hydrogen atoms with deuterium atoms in hydrogen/deuterium exchange (H/D exchange or HDX) methods, (ii) the covalent modification  of amino acid residues  in various covalent labeling workflows, and (iii) chemical cross-linking, in which two spatially proximate amino acid side chains are covalently coupled  72]. Pa. Pa72]. 6 intracellularly) and diversity in their binding affinities. Additionally, these include the variety of amino acid residues that can be targeted by cross-linkers and the decreased solubility of cross-linked protein complexes that, in turn, causes problems during digestion and the ionization of large cross-linked products ..149].The changing patterns of RNA–protein interactions is critical for cellular functions, which are then regenerated based on the subcellular localization and environment stimuli . CharactCross-linking methods can be used to identify protein–RNA interactions in vivo. To do so, the RNA is purified under denaturing conditions to remove non-covalent interactions and only cross-linked components are subsequently extracted for identification. The formaldehyde, a small and bifunctional cross-linker, can easily permeate the cells and cross-links macromolecules within 2 Å, involving the protein–RNA complexes, by creating a reversible covalent linkage . MethodsThe strength of the RNA–protein interaction should be mainly considered while choosing the cross-linking approach. RNA–protein dissociation constants vary widely, and such experimentally measured constants can be in the range of high nanomolar to picomolar concentrations. Generally, the weaker RNA–protein interactions are less likely to be captured by UV cross-linking as contrary to formaldehyde cross-linking. In addition, the UV cross-linking efficiency varies according to amino acid chemistry, whereas, in the formaldehyde cross-linking, nucleophilic lysine residues are strongly preferred and cross-linked b 149]. . 149]. HIn the proteomic analysis, most of the RNA-centric methods follow quantitative mass spectrometry workflows to detect proteins that are bound to RNA. These MS approaches can be either label-free or incorporate the use of chemical labels for detection or simply quantitation. Labeling methods can use stable isotope labels or chemical tagging of proteins in samples (can be termed as controls) and, thus, the enrichment scores can be obtained by the ratio of labeled peptides and true binding partners can be identified . LabelinThe majority of studies identify RNAs that are bound to a protein are performed by purifying the protein of interest. The most common way to achieve this is by making use of the long-known fact that the protein will chemically cross-link to the nucleic acid in vivo when hit by UV light at ~254 nm . This UVLately, the formaldehyde has been used as a cross-linking reagent for CLIP method on a double-stranded RNA-binding protein, which are, in most cases, thought to UV cross-link poorly . Methyle13C labeled form of biphenyldiglyoxal . Based on a general model of the physics (or biophysics), the MD simulation predicts the conformational dynamics or time course movements (at femtosecond resolution) of every atom from a biomolecule  that is used to assist understanding of important biomolecular processes ,177,178.Currently, significant progress is being made by pairing the cross-linking mass spectrometry with MDS in order to explore a wide range of biological questions concerning protein motions, interactions, and their assemblies. For example, Brodie et al. presented an integrative structural biology approach, in which short-distance cross-linking constraints are incorporated into rapid discrete molecular dynamics (DMD) simulations . They prRapidly growing technologies to map out interactions between protein–protein /RNA/DNA are critically important since the biological function of these molecules is extremely influenced by their structures, complex formation, locations and regulatory networks. Herein, we reviewed a suite of methods that are valuable for detecting such interactions between biomolecules of the cell. Special emphasis was on the combination of cross-linking with native mass spectrometry technique to yield mutual benefits while characterizing protein–protein, protein–RNA, or protein–DNA interactions, though, with a slightly modified approach. The CLMS strategies allow for capturing and identifying not just stable, but also transient, dynamic, and weakly bonding molecules and, thus, it emerged as the most striking example of multidisciplinary success among hybrid or integrative structural biology methods. In addition, CLMS provides inter-residue distances that can be integrated into the molecular modeling and MD simulation techniques, in order to achieve physiologically realistic quaternary (PPI) structures. Different means to combine the knowledge of chemistry with MS to analyze essential biological systems of interest were scrutinized. Several different cross-linkers are accessible with varied chemistries for the CLMS techniques and, therefore, one should consider the protein sequence of interest to determine what sort of combination of cross-linker and cleavage enzyme yield positive results. To start with one of the most commonly used cross-linker types, like amine reactive cross-linking  and carboxyl-amine cross-linking (COOH-NH2: EDC or sulfo-NHS zero spacer length), is advisable.Furthermore, we propose (or speculate) that the CLMS approach, though, not previously carried out, could be merged with novel interactomics techniques (proximity-dependent labeling by BioID or TurboID). Merging these two methods may bring spatial resolution to CLMS at a sub organelle level, because the BioID radius is estimated ~10 nm, and since CLMS mainly focuses on the proteins in the neighborhood of a given bait. These CLMS and proximal interactomics methods can be iteratively performed in living cells, i.e., the generated samples are the proximal species of a protein of interest labeled with biotin first and then cross-linked with interactors. Subsequently, perform streptavidin-biotin capture, followed by the digestion and cross-linked peptides identification of proximal interactors.Besides the outstanding practice of CLMS in cross-linked peptide identification, it is worth noting that the failure to detect cross-links or only a few cross-links is related to several factors. For example, the lengths of the cross-linked peptides are too short or long, defective fragmentation of the cross-linked peptide, the cross-linked peptide is below the MS detection threshold, or the cross-linker is too short to link the appropriate residues. A few additional factors are: the sequence of interest is not fitting well with the cross-linker chemistry concerning reactive or cleavable residues, imperfect reaction conditions, the whole amount is too low, and extraction difficulties of the cross-linked peptides from a gel . Therefore, the functional groups in a linker have to be tested and characterized thoroughly, as well as demonstrated precisely within the CLMS framework. Additionally, we addressed different protocols that are useful for digestion in CLMS methodologies, such as ‘in-gel’, ‘out-gel’, ‘in solution’, and FASP, with their successful usage. Alongside the CLMS application for identifying the protein–protein interactions, this review covers or describes the cross-linking methods for studying protein–RNA as well as protein–DNA interactions, which apparently arises as fertile ground for future CLMS utilization.