Both de novo QSAR models have a sound balance of descriptive and statistical approaches. Decidedly, these QSAR models are serviceable in the development of MDA-MB-231 TNBC cell antagonists.In the present endeavor, for the dataset of 219 in vitro MDA-MB-231 TNBC cell antagonists, a (QSAR) quantitative structure–activity relationships model has been carried out. The quantitative and explicative assessments were performed to identify inconspicuous yet pre-eminent structural features that govern the anti-tumor activity of these compounds. GA-MLR  methodology was employed to build statistically robust and highly predictive multiple QSAR models, abiding by the OECD guidelines. Thoroughly validated QSAR models attained values for various statistical parameters well above the threshold values (i.e., R Cancer is among the most clinically challenging and life-threatening ailments, globally. In 2020, more than 19.29 million new cancer cases and nearly 10 million related deaths worldwide were chronicled, of which 2.3 million are breast cancer cases with 685,000 related deaths .Triple-negative breast cancer (TNBC), the third subtype, unlike the first two, does not contain ER, PR or overexpressed HER2 protein, and this makes it hardest to treat. Therefore, chemotherapy is the mainstay for the treatment of TNBC, essentially at all the stages of breast cancer. Breast oncology is based on in vivo and in vitro studies performed against breast cancer cell lines (BCCL). BT–20 is the first BCCL long-established back in 1958. Despite of tireless work in this area, permanent BCCLs obtained have been notably low in number (about 100 only). Most of the available BCCLs are issued from metastatic tumors, mainly from pleural effusions. MCF-7, T-47D, SK-BR-3 and MDA-MB-231 are a few BCCLs well-reported in breast oncology .MDA-MB-231, an epithelial human BCCL, is known to be the most aggressive, invasive and ill differentiate TNBC cell line. Proteolytic degradation of the extracellular matrix brings about invasiveness of the MDA-MB-231 cells , urea-baIn silico lead optimization is a feasible, economical, absolutely eco-friendly, quantitatively predictive, relatively faster drug discovery approach, which is the utmost need of time. Sparse animal trials and result accustomization make computer-assisted drug designing (CADD) a pragmatic approach. QSAR is one of the prospering branches of CADD, which persistently and outstandingly contributes towards lead optimization ,24.QSAR–a cross-curricular, blended approach, ascertains mathematical correlation between structural traits of the molecule and associated bioactivity on a statistical basis. General steps in a QSAR analysis protocol are (I) selection of a sufficiently large, pertinent molecular dataset with desired bioactivity, (II) 3D structure generation and optimization, (III) molecular descriptor calculation and consequential pruning using an apt statistical method, (IV) QSAR model generation using an algorithm that furnishes propitious molecular descriptors and (V) cromulent validation of developed QSAR model(s) . DescripIn the present endeavour, a qualitative cum quantitative SAR model for a series of 219 MDA-MB-231 cell anti-proliferative compounds has been performed. The results are useful to optimize compounds for better anti-TNBC activity.Although the present study is based on the moderate size dataset, the presence of diverse molecular scaffolds, functional groups, substituents, different rings, viz. non-aromatic, homoaromatic, heteroaromatic, fused rings, spiro compounds, etc., have notably covered an enormous chemical space. Hence, both of the QSAR models generated are based on the divided dataset only.2, R2adj, CCCtr, etc., have values well above the approved threshold values, which confirms that the QSAR models are statistically acceptable with an adequate number of molecular descriptors in them. Internal validation parameters such as Q2LOO, Q2LMO, etc. have values that vouchsafe the statistical robustness of the QSAR models  + 0.013(±0.006) * all_MSA3 – 0.538(±0.198) * com_Splus_7A + 0.379(±0.265) * fHringC4B + 0.107(±0.027) * fOH5B – 0.178(±0.077) * fringNC8B + 0.924(±0.140) * com_sp2N_2A.pIC2 = 0.79, R2adj = 0.78, Q2LOO = 0.77, Q2LMO = 0.77, RMSEtr = 0.35, MAEtr = 0.27, RSStr = 21.23, CCCtr = 0.88, RMSEcv = 0.36 MAEcv = 0.28, PRESScv = 22.89, CCCcv = 0.87, R2ext = 0.72, Q2-F1 = 0.72, Q2-F2 = 0.72, Q2-F3 = 0.72.R50 = 5.072(±0.193) + 0.012(±0.006) * all_MSA3 – 0.480(±0.203) * com_Splus_7A – 0.096(±0.068) * com_don_6A + 0.101(±0.028) * fOH5B – 0.160(±0.078) * fringNC8B + 0.969(±0.121) * com_sp2N_2A.pIC2 = 0.79, R2adj = 0.78, Q2LOO = 0.77, Q2LMO = 0.76, RMSEtr = 0.35, MAEtr = 0.27, RSStr = 21.24, CCCtr = 0.88, RMSEcv = 0.36 MAEcv = 0.28, PRESScv = 22.94, CCCcv = 0.87, R2ext = 0.76, Q2-F1 = 0.76, Q2-F2 = 0.75, Q2-F3 = 0.75.R2ext and Q2-Fn are noteworthy. This highlights the importance of both of the models. Moreover, these two QSAR models differ in one variable (molecular descriptor) only, viz. fHringC4B in model 1.1 (with positive sign of the coefficient) and com_don_6A (with negative sign of the coefficient) in model 1.2. In the discussion section, we have explained the importance of these two molecular descriptors, and consequently, the importance of these two QSAR models in terms of their usability and applicability.Although for both these models, values of almost all the statistical parameters related to fitting criteria and internal validation are essentially the same, the differences in values of statistical parameters related to external validation, i.e., RR2 = 0.67, Q2 = 0.55 and R2pred = 0.53) [R2 = 0.84, Q2LOO = 0.83 and R2pred = 0.75) [Among the reported QSAR studies of MDA-MB-231 TNBC cells, the particularly recent work has been done using the dataset of 61 parthenolide derivatives ( = 0.53) , and yet = 0.75) . These QBoth models 1.1 and 1.2 have been constructed using the divided dataset only. These models differ in only one descriptor out of a total of six descriptors. Although, the change in the activity of each molecule, to a large extent, is a combined effect of all the six molecular descriptors, in the ensuing section effect of variation in each molecular descriptor on biological activity of the irrespective molecule, and is illustrated with examples see .1. all_MSA3, fHringC4B, fOH5B and com_sp2N_2A: All of these molecular descriptors have positive values of the coefficient and increase in the values of these molecular descriptors, which increases MDA-MB-231 anti-proliferative activity.all_MSA3 : The observation is supported by comparing compound 36 (IC50 = 4.746) with compound 39 (pIC50 = 5.772), for which an increase in the value of all_MSA3 from 0 for compound 36 to 13.07 for compound 39 results in an increase in pIC50 value by about 1 unit (about ten-fold increase in MDA-MB-231 cell anti-proliferative activity). Compound 23  and compound 73  is another pair used as an example to support this observation. Partial charges on each atom of the compound with the non-zero value of the all_MSA3 descriptor are shown explicitly in all_MSA3 to calculate molecular surface area takes into consideration atoms with partial charges in the range −0.099 to 0.000 only.fHringC4B (Frequency of occurrence of ring carbons which are present exactly at four bonds from the hydrogen atom): this molecular feature is absent in all the ten least active compounds but present in all the ten most active compounds : In compound 42  there are four hydrogen atoms which are five bonds away from oxygen. Whereas, in ten-fold more potent MDA-MB-231 anti-proliferative compound 41 , eight such hydrogens are present. This illustrates the significance of the higher value of the fOH5B molecular descriptor in order for leads to be better MBA-MD-231 anti-proliferative agents (e agents .com_sp2N_2A (Number of sp2-nitrogen atoms within 2Å from center of mass of molecule): Significance of this molecular descriptor can be rationalized with the fact that in all the ten least active compounds  There is no positively charged sulfur atom within 7Å from the center of the mass of molecule (com_Splus_7A = 0), rather the sulfur atom itself is absent in these ten most active compounds (pIC50 = 7.155–7.398)  b. All th as zero . (Centercom_don_6A (Number of donor atoms within 6Å from the center of the mass of the molecule): There is no donor atom within 6Å from the center of the mass of the molecule (com_don_6A=0); in fact, there is no H-Donor functionality such as N-H, O-H present in all the ten most active compounds (pIC50 = 7.155–7.398). Hence, there is great scope to say that the zero value for the com_Splus_7A and com_don_6A molecular descriptors have gained better MDA-MB-231 cell anti-proliferation potency to them. With two H-donor atoms , Compound 156  is the least active compound of the series that highlights the importance of absence of com_don_6A molecular descriptor for molecule to be better MDA-MB-231 cell anti-proliferator. Comparison of compound 97  with 148  and 95  with 107 also support the observation.fringNC8B (Frequency of occurrence of number of carbon atoms which are present exactly at 8 bonds from ring nitrogen atoms): Compound 49 (IC50 = 6.02 μM) with no such carbon atoms which are present exactly at eight bonds from the ring nitrogen atom found to be about eight-fold more potent than compound 91 (IC50 = 40.92 μM), which contain three such carbons (fringNC8B = 0). These observations mark the importance of the fringNC8B molecular descriptor. carbons . MoreovefHringC4B in model 1.1 (with positive coefficient) and com_don_6A (with negative coefficient) in model 1.2. All the relatively potent MDA-MB-231 cell anti-proliferative compounds from the present dataset with pIC50 ≥ 7 have fHringC4B = 1 and com_don_6A = 0  and all the relatively inactive compounds with pIC50 ≤ 5.043 have fHringC4B = 0 and com_don_6A > 0 . This observation signifies the importance of combined effect of the molecular features represented with these molecular descriptors, and eventually broadens the applicability of the present QSAR evaluation studies (see Two QSAR models proposed in the present study differ in one variable (molecular descriptor) only, viz. dies see .50 = 4.991, Predicted pIC50 = 6.015 by Model 1.1 and Predicted pIC50 = 5.961 by Model 1.2) leads to the increase in values of all_MSA3 and fOH5B molecular descriptors due to an increase in –CH2- groups in the molecule. Hence, both the models predicted extremely high pIC50 values for molecule 80. Experimentally, the typical non-planar, chair conformation of the cyclohexyl group due to steric reasons might have restricted inhibitory interaction of compound 80 with the target (pIC50 = 4.991), and hence compound 80 turned out as an outlier. Compound 154  consists of multiple scaffolds with different pharmacophores which (pIC50 = 7.097) might practically have increased the possible inhibitory potency of the molecule through an increased number of favorable interactions, but owing to the same reason, there is an increase in the value of fringNC8B and com_don_6A molecular descriptors (both have negative coefficient), and this leads to the extremely low pIC50 value prediction by both of the models.There are five outliers, viz. molecules 80, 154, 156, 160 and 183 (with >2.5 σ), which are revealed in the Williams plot see . Lipophi50 = 4.066, Predicted pIC50 = 5.239 by Model 1.1, Predicted pIC50 = 5.227 by Model 1.2) and compound 160  are developed from some of the structural fragments of compound 154 , presence of a Bromine (Br)-heavy atom with large atomic/ionic volume as a substituent caused shifting of the position of the center of the mass of the molecule, which reduced the value of com_sp2N_2A (positive coefficient) to 1, and hence both models predicted lower pIC50 values than was experimentally observed.The 50 = 0.04 to 86 μM) have been selected for the present work [50 values were converted to pIC50 (pIC50 = –logIC50) before actual QSAR analysis. To demonstrate the variation in bioactivity with chemical features covered by the present dataset, ten least and ten most active molecules have been depicted in 50 and pIC50 values for all the molecules are present in A total of 219 MDA-MB-231 cell antagonists having moderate anti-proliferation potency , was used to draw structures of all 219 molecules. Subsequent conversion of these structures to corresponding 3D structures was achieved using another free and open-source chemistry toolbox, OpenBabel 2.4.0. Thereafter, an optimization and molecular alignment was carried out using the force field MMFF94 available in TINKER (default settings) and Open3DAlign, respectively.A free comprehensive chemical drawing package, ChemSketch 12 Freeware , for example, is necessarily mandatory. Hence, some molecules from the dataset were randomly kept aside as a prediction set, and remaining molecules (training set) were subjected to SFS treatment to develop the QSAR model. The QSAR model(s) generated is validated using molecules in the prediction set.Random splitting of the dataset using random splitting option in QSARINS v.2.2.4 into an 80% training set (175 molecules in training set) and a 20% prediction set (44 molecules in prediction set) was achieved. The training set was used for QSAR model development, and the prediction set was utilized for external validation.ii. 2LOO as a fitness function for feature selection was deployed in genesis of the GA-MLR-based QSAR models with double cross validation. Up to six variables, there was a generous increase in the Q2LOO value, but minor augmentation was observed thereafter. Consequently, the selection of the molecular descriptor was confined to a set of six descriptors to foil the danger of over-fitting, and this additionally helped to derive easy and informative QSAR models  with best values of these parameters and with best predicative ability (Q2-Fn > 0.71) were selected.Abide by OECD guidelines; for ensured proper validation, all the models were subjected to internal and external validation, Y-randomization and model applicability domain (AD) analysis using QSARINS 2.2.4. Robustness of the GA-MLR-based QSAR model was adjudicated on the basis of (a) internal validation based on Leave-One-Out (LOO) and Leave-Many-Out (LMO) procedure; (b) external validation; (c) Y-randomization and (d) fulfilling of the respective threshold value for the statistical parameters: RThe contracted molecular descriptor set is a heterogeneous set of descriptors in the sense that it comprises 0D to 3D descriptors, charge descriptors and molecular properties, etc. that have covered an adequately comprehensive descriptor space. Subjective feature selection (SFS) in QSARINS v2.2.4 was executed in order to construct statistically robust GA-MLR-based QSAR models. Thereafter, thorough statistical validation , Y-randomization and applicability domain analysis of the derived models was done, abiding by OECD ,42,43,442ext and Q2-Fn, resulted in extremely high values for both models. Molecular features which appeared in QSAR models, such as an increased number of four bond-distant ring carbons from hydrogen, five bond-distant hydrogen from oxygen, a lesser number of eight-bond distant carbons from ring nitrogen, etc., are easy to incorporate in a manner that will make optimization easy and scopeful. These models will help in optimizing present compounds to more potent MBA-MD-231 anti-proliferative leads to treat triple-negative breast cancer.A well-founded balance of statistical QSAR and descriptive QSAR with highly precise bioactivity predictability  on incorporation of molecular features is furnished by both models. Various statistical parameters that are indicators of preciseness of the external predictability, especially R


Intervertebral disc degeneration (IDD) has caused great trouble in people’s lives. Dysregulated long noncoding RNAs (lncRNAs) are closely linked to IDD progression. Our study aims to analyze the role of LINC00917 in the progression of IDD. Forty nucleus pulposus (NP) IDD tissues and 40 NP tissues of intervertebral discs without apparent degeneration were collected. TBHP was used to induce IDD. Cell proliferation was measured using the MTT and EdU assays. Pyroptosis was detected using flow cytometry. RT-qPCR and Western blot assays were performed to determine mRNA, miRNA, and protein expression. Dual-luciferase reporter and RNA pull-down assays were performed to verify the relationship between LINC00917 or NLRP1 and miR-149-5p. LINC00917 expression was enhanced in TBHP-treated nucleus pulposus cells (NPCs). The knockdown of LINC00917 promoted proliferation and inhibited cytotoxicity, inflammatory response, and pyroptosis of NPCs. LINC00917 functions as a sponge for miR-149-5p. Having silenced miR-149-5p, the effects of LINC00917 knockdown on NPC proliferation and inflammation-induced pyroptosis were alleviated. NLRP1 overexpression induced cellular dysfunction and pyroptosis of NPCs. LINC00917 knockdown restored NPC cellular functions and inhibited IDD progression by modulating the miR-149-5p/NLRP1 axis.  However, the existing side effects neutralize the clinical outcomes. Therefore, it is important to explore an effective method for treating IDD.In general, intervertebral disc degeneration (IDD) induces the development of lower back pain and shoulder and neck pain . IDD is IDD is usually caused by programmed cell death of nucleus pulposus cells (NPCs), because NPCs are essential for maintaining the mechanical and biochemical homeostasis of the intervertebral disc . Recent In recent years, the potential of long-chain non-coding RNAs (lncRNAs) for IDD treatment has attracted increasing attention ,15. LncRTherefore, in this study, we predicated and confirmed that LINC00917 functions as a miR-149-5p sponge and NLRP1 is targeted by miR-149-5p via bioinformatic tools. We aimed to explore the specific action mechanism of LINC00917 in IDD. In addition, we hypothesized that LINC00917 participates in the proliferation and pyroptosis of the TBHP treated NPCs via regulating miR-149-5p/NLRP1 axis.http://starbase.sysu.edu.cn/). Besides, the target gene of miR-149-5p was predicted in online TargetScan software (http://www.targetscan.org/vert_71/).The target miRNA of LINC00917 was predicted by in online starBase software  patients  and 40 NP tissues of intervertebral disc without apparent degeneration patients  were collected from idiopathic scoliosis surgery in Air Force Medical Center of PLA. All patients agreed to our experiments and signed the informed consent form. Our research was approved by the ethics committee of Air Force Medical Center of PLA.3 pieces, and placed them in a centrifuge tube. Trypsin (0.25%) was added to the tubes for digestion at 37°C in water and centrifuged for 20 min. Next, the supernatant was discarded and type-II collagenase was added. NPCs were maintained in the DMEM-F12 medium supplemented with 10% FBS and 5% CO2 at 37°C. To establish the IDD model in vitro, the NPCs were cultured with 100 μM TBHP for 4 h. NPCs from the control group were added to the same amount of culture medium.Next, according to the previous study , we collCells in the logic growth phase were transfected with siRNA targeting LINC00917 , miR-149-5p mimic and inhibitor, overexpression NLRP1 (oe-NLRP1), and their controls  using Lipofectamine® 3000 reagent . si-LINC00917 1#: 5’-CUUUCUGUCACAUUGACCACCCUG-3’; si-LINC00917 2#: 5’-UAGCACCAUUUGAAAUCAGUGUU-3’.−ΔΔCt method was used to quantify the relative expression according to a previous study [TRIzol  was used to separate RNA. Nanodrop1000  was used to analyze the purity and concentration of the extracted RNA to ensure 8 ≤ OD260/OD280 ≤ 2.0. The diluted RNA was reverse-transcribed using a reverse transcription kit . The reverse transcription parameters were as follows: reaction at 37°C for 15 min, 3 cycles; denatured at 85°C for 5 s. Next, the SYBR® Premix Ex Taq™ quantitative kit was used to perform RT-qPCR. The reaction conditions were as follows: pre-denaturation at 95°C for 30s, 40 cycles; denaturation at 95°C for 5 s and annealing at 60°C for 30s. GAPDH was used as the internal reference. The 2us study .4 cells/well and cultured for twenty-four hours. Then, the cell was treated with MTT for four hours. Next, the cells were treated with dimethyl sulfoxide. Finally, optical density was measured at 490 nm using a microplate reader .According to a previous study , NPCs inAccording to a previous study , NPC cel6 cells/ml) were seeded into 24-well plates. The cells were then washed and resuspended. After that, the cells were incubated with 5 ml μl Hoechst33342 and 5 μl PI in the dark for 15 min. Finally, the pyroptotic cells were analyzed by flow cytometry .According to a previous study , NPCs  were intravenously injected via the tail vein into designated group to knockout the LINC00917 expression. After four weeks, the mice were euthanized and intervertebral disc tissues were collected. The animal studies were approved by the ethics committee of Air Force Medical Center of PLA.Before the mice in each group were killed, the mice were placed in a transparent observation box and counted with a stopwatch. From the contact between the hind foot and the tray to the emergence of tiptoe, retraction, licking and struggle, the latency of hot stimulation foot contraction reflex was recorded, and paw withdrawal latency and paw withdrawal threshold were measured. Additionally, the BBB score were also recorded according to a previous study .According to a previous study , all micAccording to a previous study , the LDHThe data were analyzed using SPSS 21.0. The Student’s t-test was used to analyze the differences between the two groups. One-way analysis of variance was performed for comparisons between multiple groups. The results are expressed as mean ± SD. Where p smaller than 0.05 means that the difference is statistically significant.In this study, LINC00917 expression was up-regulated in IDD tissues and TBHP treated NPCs. However, knockdown of LINC00917 promoted the proliferation, and inhibited the inflammatory response and pyroptosis of NPCs via regulating miR-149-5p/NLRP1 axis.First, we demonstrated that LINC00917 levels were dramatically elevated in the IDD tissues ). In addNext, after transfection with si-LINC00917 1# and si-LINC00917 2#, LINC00917 expression was dramatically downregulated in NPCs ). AccordUsing online starBase software, miR-149-5p was predicted to target LINC00917 ). The biTo analyze the specific mechanism of LINC00917 in IDD, we designed and transfected miR-149-5p inhibitor and oe-NLRP1 into NPCs. After transfection, miR-149-5p levels were depleted and NLRP1 levels were enhanced . MTT andLast, we explore the role of LINC00917 in the IDD mice. The results of HE staining showed that in the sham group, the nucleus pulposus tissue of the mice was normal. In the Model group, the nucleus pulposus tissue is deformed, the fibrous rings are arranged disorderly, and the matrix rich in mucopolysaccharide and cell clusters penetrate each other. These phenomena were alleviated after LINC00917 knockout ). BesideIn the current study, LINC00917 expression was upregulated in IDD tissues and TBHP-treated NPCs. However, the knockdown of LINC00917 promoted proliferation and inhibited the inflammatory response and pyroptosis of NPCs by regulating the miR-149-5p/NLRP1 axis.Pyroptosis is inflammatory programmed cell death mediated by two cysteine aspartate proteases (caspases): classical Caspase1 and non-classical caspase4/5/11. There are five inflammasomes that can stimulate pyroptosis: NLRP1, NLRP3, NLRC4, IPAF, and AIM2 of the NLR family. The classical pyroptosis pathway is mainly induced by the activation of caspase1, which is activated by the NLR family inflammasome . ActivatRecently, the role of lncRNAs in the development of IDD has attracted extensive attention. Accumulating evidence has demonstrated that significant changes occur in the expression of many lncRNAs in IDD. Wan et al.  confirmelncRNAs can function as sponges” for miRNAs to participate in the regulation of the pathophysiological processes of IDD; that is, lncRNAs play a competitive role in endogenous RNA by targeting miRNAs. Zhang et al.  found thmiRNAs participate in IDD progression by binding to the 3’ UTR of related genes. For instance, miR-623 suppresses oxidative stress-induced apoptosis of NPCs by binding to thioredoxin-interacting protein . MesenchTo sum up, LINC00917 inhibits the proliferation, induced the inflammation and pyroptosis of NPCs via targeting miR-149-5p/NLRP1 axis. This study provided potential therapeutic targets for IDD.


This article discusses the properties of as many as 30 carbene–ZnX Undoubtedly, carbenes belong to one of the most important groups of compounds in both organic and organometallic chemistry ,16,17,18p-orbital perpendicular to this plane. Importantly, this singlet state is stabilized by the simultaneous presence of several important factors. First of all, the presence of nearby nitrogen atoms is crucial, which stabilize the singlet state through both the inductive p-orbital of the CIn the singlet state, the divalent carbene carbon atom has a lone electron pair in the ring plane and an unoccupied  arrows) ,21,22,23aracter)  and a smaracter) ,25,26. Iaracter) . A very aracter) .p orbital on the carbene carbon atom can cause, depending on the situation, the carbene molecule to exhibit its electrophilic [The simultaneous presence of a lone electron pair and the formally empty rophilic ,31,32,33rophilic ,16,17,18rophilic ,17.The enormous variety of NHC-M systems makes NHCs critical in organometallic catalysis ,36,37,38tert-butyl  are of increasing practical importance ,62,63,64ns as N4 . For thitert-butyl  , IDipp–Z(DEDHUQ) , IDipp–Z(YIDQIN) ). The mainc bond ) will beGeometries of all the systems were fully optimized on the MN15/6-311+G(d) level of theory utilizing Gaussian 16 . There wtert-butyl , it actually concerns the entire dimer, i.e., it is a global quantity, and only when the described interaction is dominant can the determined dissociation energy describe this interaction . The disIt is somewhat surprising that despite such diverse R substituents and different X groups, the range of C⋯Zn distances is very narrow, from ca. 2.12 Å  is the delocalization index . Its valIn full agreement with the result based on ρC⋯Zn , the valWhen discussing the results based on QTAIM, it is worth mentioning that in addition to structural symptoms, the presence of the previously discussed interactions accompanying the leading zinc bond is also suggested by the presence of appropriate bond paths on molecular graphs. Some of the more interesting and representative examples are shown in As can clearly be seen, in the case of the IMe–ZnHIt is not unusual that molecular graphs suggest many different long-range interactions through the presence of appropriate bond paths. This is especially true in the case of larger molecular systems with a high concentration of functional groups. A good and quite clear example is the molecular graph of the IDipp–ZnHAs one can see, the IR–ZnXen shown ,103,104 Parameters based on QTAIM are often local as they relate to specific critical points on the molecular graph and are in a way blind to what is happening in the immediate vicinity of these points. One way out of this limitation is the NCI method ,76, whicof atoms . To furts isosurface, suggesting that the interactions are relatively strong . In the case of the latter of these complexes, it is clearly visible that the C-H⋯H-C dihydrogen interaction should be much weaker, but also stabilizing (green). The case of the IAd–ZnEtAs already mentioned , the IMeAs discussed earlier see , the comThis article discusses the results of research on the properties of as many as 30 complexes of the IR–ZnXIt has been shown that the strongest C⋯Zn bond should be present in IMes–ZnHIt has been shown that the twist angle of the ZnX


As a refence, we also study the properties of SAMs of electron saturated 1-dodecanethiol  molecules. The stability of the considered SAMs before and after electron-irradiation is studied using low energy Ar+ cluster depth profiling monitored by recording the X-ray photoelectron spectroscopy (XPS) core level spectra and the UV-photoelectron spectroscopy (UPS) in the valance band range. The results indicate a stronger mechanical stability of BPD SAMs than the C12 SAMs. The stability of BPD SAMs enhances further after electron irradiation due to intermolecular cross-linking, whereas the electron irradiation results in deterioration of C12 molecules due to the saturated nature of the molecules. The depth profiling time of the cross-linked BPD SAM is more than 4 and 8 times longer than the profiling time obtained for pristine and BPD and C12 SAMs, respectively. The UPS results are supported by density functional theory calculations, which show qualitative agreement with the experiment and enable us to interpret the features in the XPS spectra during the etching process for structural characterization. The obtained results offer helpful options to estimate the structural stability of SAMs which is a key factor for the fabrication of molecular devices.The stability of the molecular self-assembled monolayers (SAMs) is of vital importance to the performance of the molecular electronics and their integration to the future electronics devices. Here we study the effect of electron irradiation-induced cross-linking on the stability of self-assembled monolayer of aromatic 5,5′-bis(mercaptomethyl)-2,2′-bipyridine [BPD; HS-CH In this field, the molecular self-assembly is  becoming a standard tool in nanofabrication, which enables one to create interface structures with enhanced functionalities. Self-assembled monolayers (SAMs) of thiol-end molecules on noble metal surfaces are promising hybrid systems for practical applications in electronics due to high stability and outstanding electronic and transport properties. Such systems are also suitable for large scale production due to easy preparation, materials availability, and large surface coverage at low cost.The saturation in electronics devices performance is due mainly to the physical limitation of the silicon technology in the nanodomain. Molecular electronics, implementing organic molecules as building blocks, has a great potential to substitute the silicon technology at the nanoscale due to exceptional physical and chemical properties that molecular systems can provide4. Depending on the molecular backbone, a different anchor angle between the sulfur and gold atom is realized, which further determines the orientation of the SAM arrays. X-ray photoemission measurements also reveal a binding energy shift with different bonding positions of thiol on Au (111) single crystal surface5. Studies on the effects of the substrate morphology on the adsorbed molecules have been conducted in terms of, molecular size, charge exchanging, electric dipolar modification, working function variation and electronic level alignment to the Fermi edge. For example, tuning the work function of a metallic substrate with a molecular electric dipole involves attaching a polar head group to the SAMs, varying the dipole of the binding group at the interface, or inserting a polar group into the molecular backbone7.To understand the properties of the molecular systems and further improve their performance, extensive research has been conducted addressing problems ranging from the molecular architecture to the organic/inorganic hybrid interface formation. A particular focus has been given to the effects of the thickness of the SAMs, by using different types of head groups/binding groups and different molecular backbone from alkyl chain to π-conjugated aromatic structures. Thiol-gold interfaces, through S–Au covalent bonding, are of fundamental importance in SAM creation and therefore, their properties are studied using different measurement techniques, such as X-ray photoelectron spectroscopy (XPS) and scanning tunneling microscopy (STM). It has been assumed that such chemical thiol-gold bond is formed by breaking S–H bonds near the interface9. For example, Taucher et al. used DFT in describing chemical vs. electrostatic shifts in XPS spectra of organic SAMs10. In another study, Cabarcos et al. investigated the influence of embedded dipole layers on the electronic properties of alkanethiolate SAMs using different experimental techniques, such as infrared reflection absorption spectroscopy (IRS), high-resolution XPS, ultraviolet photoelectron spectroscopy (UPS), atomic force microscopy (AFM), and Kelvin probe (KP) AFM11. The experiments were complemented with DFT-based electronic band-structure calculations to get a fundamental insight into the processes taking place in the considered systems. First principles calculations have also been successfully implemented to interpret the experimental results in studying the effect of embedded dipole moments of molecules, for interface engineering through self-assembly12. Recently, Berdiyorov and Hamoudi used first-principles calculations to study the effects of molecular backbone and anchoring groups on the transport properties of aromatic molecules sandwiched between metallic electrodes14.Density functional theory (DFT) is known to be an effective predictive tool in studying the formation process and properties of SAMs on metallic surfaces, and interpreting the results of XPS measurementsIn this work, we present a detailed study of structural and electronic properties of SAMs of aliphatic 1-dodecanethiol (C12) and aromatic 5,5′-bis(mercaptomethyl)-2,2′-bipyridine (BPD) molecules on Au (111) substrate. Well-ordered molecular arrays are obtained as revealed from the S2p core-level XPS spectra. To investigate the interface properties in detail, we apply a low energy cluster-type depth profiling method, and the system evolution is monitored by XPS on both core level of C1s and S2p, and by UPS in the valence band range. Such measurements enable us to test the stability of the molecular structures by peeling-off the molecular fragment gradually from the top of the SAMs towards the interface using atomic collision techniques. We also study the effect of electron beam irradiation on the resistance of the molecular SAMs to the etching. The stability of the aromatic molecules increases by more than 4 times due to electron irradiation-induced cross-linking of the aromatic BPD molecule. On the contrary, the stability of the C12 molecules decreases after electron irradiation. We further conduct DFT calculations to describe the phenomenon of the binding energy shift during the etching process.15. Since the BPD attached to the Au surface is oriented with a stand-up structure, the signal from the S–Au at the interface is attenuated by the molecular backbone. This is not the case for the –SH signal as it locates on top of the monolayer and photoelectrons reach the vacuum without any attenuation. This explains the relatively higher –SH signal than the S–Au despite the equal number of sulfur atoms in the BPD structure. Due to the different nature of the molecules, the C1s spectrum of the BPD molecules is also different from the one obtained for C12 molecules. The component at 284.8 eV arises from the C–C moieties, and C=C in the ring while a strong shoulder at higher binding energy of 285.6 eV was attributed to C–N signal17 .SAMs of both C12 and BPD molecules were characterized by XPS before and after the electron irradiation through the S2p and the C1s core level spectra see Fig.  with a p5. Briefly, molecules form dense SAM due to non-bonding intermolecular interactions to provide minimum space packing between the molecules. However, electron irradiation leads to intermolecular cross-linking, i.e., it generates bonding between the molecules due to the electron attachment/detachment phenomenon. This will reorganize completely the topology of the molecule at the interface, leading to the decrease of the intermolecular spacing and create new adsorption sites for the thiol on Au at the second adsorption position. The relative intensity of the 163.5 eV component is slightly reduced when referenced to the 161.9 eV component.We then test the film resistance to the electron irradiation. The electron beam was set 1000 eV in energy with an exposure time of 30 min on top of the two SAMs. A direct comparison of the C1s and S2 spectra before and after electron irradiation is shown in Fig. S2 in the supporting information. It is seen from this figure that C1s signal of the C12 SAM exhibits a great loss in intensity  and the S2p signal gets distorted both in intensity and the profile. On the contrary, in the case of the BPD SAM the effect of the electron irradiation on both C1s and S2p spectra is much less pronounced; the reduction in the signal amplitude is less than 10% . We specifically clarify such evolution by looking into the S2p deconvolution. As shown in Fig. + cluster ions incident at 45° to the surface and by recording both the UPS and the C1s/S2p core level spectra. Figure + clusters enabled the removal of molecular fragments from the top of the molecules gradually, leading to the intensity of the C1s attenuating as a function of the etching time. Such attenuation turned out to be more pronounced in the case of C12 molecule than BPD by comparing Fig. + beam with the same energy: while it needs 120 s of etching on the pristine BPD SAM  is important characteristics for molecular systems as it determines the performance of the molecular devices working under similar environment. The modification of the SAMs induced by the irradiation has been reported, including decomposition of the SAM’s entitiesAs we have mentioned above, at low energies (0–7 eV) the UPS features from the underlying alkyl chain of the C12 molecules and aromatic parts of the BPD molecules are gradually replaced by the photoionization features of the gold substrate as the length of the molecules decreases. This effect of the substrate is more pronounced in the case of C12 SAM. The position of the maxima of the UPS spectra does not change with etching in this range of the energy. In the region between 12.0 and 17.0 eV we see decreasing in the intensity in the background of scattered secondary electrons that rise initially from photoemission in the alkanethiol layer, as well as in the gold near-surface region. In both cases, we observed a clear shift to low binding energy in the cutoff energy region between 12.0 eV and 17.0 eV as the molecular length decrease.hv, the cutoff and fermi level Ef was measured to obtain the work function Φ with the following equation:To explain this shift, we first calculate the work function (Φ) by looking at the cutoff and Fermi level in the UPS spectra during the etching process. We picked out the simulated spectra of C12, C8 and C4 for the C12  and an aromatic dithiol (BPD) molecule self-assembled on gold (111) surface is examined using low energy Ar35 as follows. C12@Au SAM was prepared using 1 mM solution of C12 in absolute ethanol. The solution was degassed for 30 min before immerging the gold substrate. The system was kept for 4 h under reduced light condition and nitrogen blanket. After that, the gold substrate was washed three times with absolute ethanol and dried with nitrogen gas. BPD@Au SAM was prepared by using 1 mM solution of BPD in hot hexane (60 °C). The solution was maintained at 60 °C and degassed for 30 min before immerging the gold substrate. The system was kept for 1 h, under reduced light condition and nitrogen blanket. After that, the gold substrate was washed once with hexane and three times with absolute ethanol, then dried with nitrogen gas. Surface analysis of the studied SAMs were performed immediately after the sample preparation, to avoid oxidation of the adsorbed molecules.All the chemicals and solvents were purchased from Sigma Aldrich and used for sample preparation without further purification. Pure ethanol and hexane were degassed under nitrogen gas flow for 30 min prior to their use. Gold (111) on mica substrates were purchase from PHASIS Switzerland, rinsed with absolute ethanol and dried under nitrogen gas flow prior to use. SAM structures are created following the procedure described in Ref.The photoemission measurements were performed on the standard Thermo Fisher ESCALAB 250XI type XPS platform. A monochromatic Al Kα Anode X-ray beam of 1486.6 eV was used with an energy resolution of 0.5 eV. The XPS spectra were obtained with a normal emission and a beam incident angle of 45° to the surface normal. All the energy values were calibrated with respect to the Au4f located at 84.0 eV. UPS measurements were conducted with a He discharge lamp with a beam energy of 21.2 eV. A perpendicular angle was applied for data acquisition and bias was given for the cutoff measurement. UPS values were calibrated to the Au Fermi level.+ cluster that enables a gentle etching of the molecular layer at atomic level. The etching beam arrives at the surface at 45° to the surface normal. The ion beam was set at 2000 eV that evenly distributed on a total cluster group of 2000 Ar+ ions, with a beam current of 10 µA. The electron irradiation exposure of the sample is performed by the Reflective electron energy loss spectroscopy (REELS) mode with an electron beam of 1000 eV. The dose density is evaluated by a faraday cup and a value of ~ 70 × 10–7 C/(cm2 s) is calculated. The exposure time was 30 min.The depth profiling was performed with a MAGCIS type ion gun. The procedure was conducted with a low energy Ar36 to account for the exchange–correlation energy. The van der Waals interactions were taken into account using Grimme’s DFT-D3 empirical dispersion correction to the PBE37. The projected-augmented wave (PAW) formalism was employed to treat the core-valence electron interactions. The valence states were expanded in a plane-wave basis set with an energy cutoff of 500 eV. A k-point sampling of 6 × 6 × 1 was used for the Brillouin zone integration38. The calculated density of states was broadened by a Gaussian width of 0.4 eV. All calculations were performed using the Vienna Ab initio Simulation Package (VASP)39. In order to calculate and visualize the electron density of a given state , we first performed a single point calculation. The states corresponding to a given energy value were then identified. Partial charge and electronic density for selected states were calculated and converted to cube format to be plotted with the VESTA package40.Structural optimizations and electronic structure calculations were performed using DFT within the generalized gradient approximation of Perdew-Burke-Ernzerhof (PBE)Supplementary Information 1.


In Europe, the antimicrobial use (AMU) for food‐producing animals has decreased rapidly. However, studies indicate that a too strict policy, with too restrictive AMU, is potentially problematic for veterinarians because it threatens animal welfare and creates tensions between farmers and veterinarians. The AMU in Sweden is among the lowest in Europe, and regulation of AMU in farm animals is strict. The aim of our study was to explore how Swedish veterinarians describe the relations between (1) being restrictive with antibiotics due to the risk of AMR and (2) concerns for animal welfare and/or the veterinarian‐client relationship.Semi‐structured interviews with 21 veterinarians, working with dairy cattle, were performed. The transcripts were analysed, and a number of dominant patterns which recurred in all, or most of, the interviews were identified.The interviewed veterinarians described AMR prevention and tackling the threat AMR poses towards public health, as central for their profession and as influencing their everyday practice and decisions on AMU. Importantly, veterinarians described accounting for AMR in everyday practice as fairly unproblematic, both in relation to animal welfare as well as in relation to farmers. The veterinarians generally perceived that they could treat animals with antibiotics when justified, and being restrictive with antibiotics was described as an expression of professional skill and not as challenging as animal welfare. Moreover, they stated that restrictive AMU seldom or never caused conflicts with farmers.Strict AMU policy and restrictive AMU do not necessarily put veterinarians in a problematic position where they are caught between conflicting demands and risks. Antimicrobial resistance (AMR) has made antimicrobial use (AMU) in farm animals a public concern and a matter in need of governance.The AMU in Sweden is among the lowest in Europe. In addition, the most used antibiotic substance is penicillinPrevious studies report that veterinarians in general are well aware of AMR and that they see themselves as having responsibility for protecting public health against AMR.Importantly, studies indicate that when veterinarians need to balance the more abstract AMR risk against current and more salient risks and interests, the latter tend to be prioritised. In a previous study,Previous studies raised questions concerning how Swedish veterinarians perceive the comparably strict AMU regulation and low AMU – do they feel that the restrictive use is in conflict with their obligation to secure animal welfare, and how does the strict AMU policy influence their relation to farmers? Interestingly, an interview study of Swedish dairy farmersThe aim of this study was to explore Swedish veterinarians’ perspectives on AMU for dairy cattle in relation to different risks and/or demands. Specifically, we aimed to explore how veterinarians describe the relations between (1) being restrictive with antibiotics due to the risk of AMR and (2) concerns for animal welfare and/or the veterinarian‐client relationship.Recruitment of veterinarians was performed through calling veterinary practices and sending further information to veterinarians who had shown an interest in participating. Participants were informed that participation in the study was voluntary and that data would be anonymized. Eventually, 21 veterinarians were recruited  due to the COVID‐19 pandemic. The interviews took between 45 min and 1.5 h. Interviews were semi‐structured and contained both questions on diagnostic practice, decisions on AMU and more general questions about AMU and AMR. The semi‐structured interview meant that an interview guide was used but that the exact content of each interview differed slightly. The interviews were recorded and transcribed by the first author. Data were stored in a storage platform. The interviews were performed in Swedish; the authors translated the quotes cited in the result section after the analysis.The analysis was performed manually. First, all interviews were read, and an initial descriptive codingThroughout the interviews, AMR and prevention of AMR were described as something the veterinarians were well aware of and as an important concern for them as professionals. Most veterinarians described AMR and careful AMU as something that they perceive as prioritised among Swedish veterinarians in general. The more junior veterinarians stated that AMR and the connection between AMU and AMR had been a central topic in their education.Veterinarian: Well, if you consider the actual development of resistance, I definitely believe that, I could phrase it like this, that it is an attitude, I guess, within the Swedish veterinary profession, because we are really good at being careful with antibiotics. .Interviewer: But how is it that, do you think, that you as a veterinarian, that you feel that responsibility, like, that you feel that, well, that you feel that you have to account for that?Veterinarian: Well, they repeated it throughout the education, that probably matters. (…) But also that, I feel that I cannot enter any veterinarian forum without AMR being addressed in one way or another. .A recurring theme in the interviews was that veterinarians described that their concern for AMR influences their everyday professional practice. Thus, their concerns for AMR were not only conceptual but had consequences for their decisions on antibiotics.Interviewer: Is it, would you say that  is present as a factor, well, when you presc…when you consider antibiotics? That you sort of… if the problems with resistance did not exist… do you believe that you would prescribe more?Veterinarian: Yes, indeed! Then it would have been really easy to treat all cows with sub‐clinical mastitis with penicillin… Everybody with a fever would get antibiotics. (…) I would mess around with Baytril (a quinolone). Everyone, everyone lying down and that has a bad udder gets Baytril if I do not have to care about resistance. .When asked about their main concerns related to AMR, most veterinarians described the threat AMR poses to human health as most central for them. While some veterinarians referred to a combination of human and animal health as their main concern, none of the interviewed veterinarians described the risk AMR poses for animal health as their only concern. Thus, throughout the interviews, the veterinarians described themselves as having a professional responsibility for the health of the future human population.Interviewer: One last question, if you think like this, what do you believe is most central for a responsible use of antibiotics for the cows? What is most important for you?Veterinarian: Yes, but that is actually our… It is human health, it really is, and then also we do not want increasing resistance. It will affect the cows as well if there is resistance, at first, but in the next step it can affect humans as well.The veterinarians were in general well aware of existing national AMU guidelines. These guidelines were described as credible and useful.Veterinarian: The district veterinarians (government organisation) in general have our treatment guidelines and such that we take into account.Interviewer: Do you use them in your daily work?Veterinarian: Yes I do. They are based on science and proven experience; it is our skilled head veterinarians that compile them so I use them a lot. The more experienced veterinarians (>10 years), however, commonly said that the guidelines were not something that they needed to read on a daily basis, but they described that their AMU in general was in accordance with the guidelines and that they sometimes read the guidelines to become updated.E. coli mastitis and that anti‐inflammatory drugs and frequent milking were more effective.A recurring theme in our interviews was that the veterinarians described a restrictive use of antibiotics and adherence to AMU policies, as aligning well with good veterinary care and animal welfare. As an example, several of the interviewed veterinarians described how the use of quinolones had been restricted by the authorities, in order to save the quinolones for human use. This policy change was referred to as something that the veterinarians supported. Central for this support was however that the veterinarians were convinced that quinolones had no significant effect on Veterinarian: And, also, enrofloxacin or Baytril, we did use that for coli mastitis, but that is also broad spectrum; it should not be used. And new results have shown that it is not effective for coli mastitis. .Importantly, the veterinarians described that they could effectively treat animals in need of antibiotics:Interviewer: Well, consider this, generally speaking: Do you feel, in your practice, do you ever feel constrained by the somewhat restrictive antibiotic policy in Sweden? Do you feel constrained by this?Veterinarian: No, I actually do not think so. No.Interviewer: You never feel like… well, feel like, this animal… that it can be at the expense of good veterinary care?Veterinarian: No, actually, no I do not think so. I think, not regarding antibiotics. In relation to other aspects, I might lack treatment alternatives and so on, but in regard to antibiotics I feel that, I think that is not a problem. It is very seldom that I feel… It has not even crossed my mind that 'oh God, what if I had that kind of antibiotics, in that case…' No I think they… No I do not think that.Interviewer: And you feel that you can treat the ones that need it?Veterinarian: Yes, I think so, I think so. With good results. .Previously in this interview, this veterinarian described that she almost exclusively used penicillin when treating cows. However, she did not experience that this prescribing practice was at the expense of good veterinary care or effective treatment. Important to note, however, is that Sweden has a favourable AMR situation with most Gram‐positive mastitis pathogens still susceptible to penicillin, which is the first‐hand choice of treatment when such bacteria are suspected in mastitis cases.Being careful with antibiotics was by several veterinarians framed in terms of professional pride and as opposed to taking  short‐cuts. They describe that use of narrow‐spectrum antibiotics, and finding other treatment alternatives than antibiotics, often require precise diagnostics and veterinary expertise.Veterinarian: Yes, for me it is also a matter of prestige, to treat what you actually know with…rather, how you could frame it, precision. To close your eyes and aim widely/broadly, then, anyone can do that. .In several interviews, being restrictive with antibiotics – and accounting for AMR, was thus framed as compatible with effective treatment in terms of certain forms of prescribing  and other kinds of treatment of animals with signs. A non‐restrictive prescribing was instead framed as an expression of lack of skills and taking professional shortcuts, rather than a way to protect animal welfare.Restrictive use of antibiotics was generally not described as problematic in relation to farmers. Throughout the interviews, the veterinarians described conflicts with farmers related to AMU and farmers that explicitly demand antibiotics, or certain kinds of antibiotics, as uncommon.Interviewer: How…you said that in general, you, you and the animal owners agree, is that also true in regard to antibiotic prescribing? Do you usually agree when it is needed and not needed?Veterinarian: Yes.Interviewer: Has it happened, that you disagree?Veterinarian: Actually, I cannot think of any situation when it has happened, so I guess I can say no .Interviewer: And how do you, how do you think, if you say that 'no, we should wait, I want to perform a culture' or…How do you think they (the farmers) would react?Veterinarian: I think they are quite well‐behaved around here. I entered a pretty good group of younger colleagues who has… paved the way for me to get…Interviewer: So, it is okay to say no? (to antibiotics)Veterinarian: Yes, I think so, I have never experienced that someone has, you know, become angry, or tried to convince me. No, no. .Veterinarian: but I still think that, I think that the farmers are like the public, everyone is aware of resistance and that, when you talk about, so most buy it and understand it. Like, it is the stuff we want to save for human healthcare. .As in these examples, the veterinarians generally described farmers as well‐educated concerning when antibiotics are needed. Thus, they stated that they are mostly called out to farms when antibiotic treatments are actually justified, and this decreases the risks of conflicts. Several of the more experienced veterinarians described that this state of affairs is a consequence of having worked with the same farmers for many years. The long‐standing relationships have not only led to farmers knowing when antibiotics might be needed but also to a more general trust in the veterinarian. In addition, the veterinarians described farmers as well aware of AMR and concerned by the risk AMR poses for human health. Thus, veterinarians described that they are able to practice and maintain restrictive antibiotic use with little resistance from clients.The aim of this study was to explore Swedish veterinarians’ perspectives on AMU in dairy cattle, in relation to different kinds of risks and/or demands. The analysis shows that interviewed veterinarians described AMR prevention and tackling the threat AMR poses to public health through restrictive AMU, as central for their profession. Similar findings have been made in other studies outside Sweden.Most importantly, our study showed that animal welfare, in terms of efficient treatment generally aligned well with being restrictive with antibiotics according to the veterinarians. This finding should be seen in the context of an already existing focus on preventive animal healthcare leading to low disease prevalence, low AMU and good welfare. The restrictive AMU policy is hence not perceived as overly restrictive but rather as preserving the effectiveness of antibiotics. A dominant pattern in the interviews was that the veterinarians felt they could combine their professional duties regarding precise diagnosis and efficient treatment, with their duties towards future human health. Thus, while previous studies reported that accounting for AMR and accounting for animal welfare in practice become opposites, our study showed that it can be different and that AMR prevention and animal welfare can actually be aligned.Our results complement a previous Swedish studyThe finding that the veterinarians saw professional AMU guidelines as credible and usable, that they described themselves as loyal to the general Swedish policy on AMU, partly differs from findings in other countries. Studies indicate that AMU guidelines tend not to be used by veterinarians or at least that guidelines are not a key source of information for veterinarians when prescribing.In this study, we have reported on the most dominant patterns identified in the interviews. There are statements that diverge from these patterns. These will be analysed further and addressed in future work. The main limitations of the current study can be related to the sample of veterinarians and the methodology. All the interviewed veterinarians were educated in Sweden, and it is possible that veterinarians educated in other countries may have other perspectives on AMU and AMR. In addition, it is possible that the veterinarians, when they self‐report on their practice, exaggerate their concern for AMR and how it influences practice. Moreover, it might be that the veterinarians who chose to take part in the study are more engaged in AMR and AMU than the average Swedish veterinarian. This limitation, however, also applies to previous studies on the subject. We argue that the qualitative interview study has important strengths. It can produce complex and detailed knowledge which cannot be retrieved thorough for example a survey study. In order to gain more knowledge on veterinarians’ AMU practices both qualitative studies, drawing on ethnography and participant observations, and quantitative audit studies of prescribing practices would be beneficial.Our results indicate that several separate and partly interrelated factors mean that restrictive AMU for dairy cattle is a fairly unproblematic matter of course in Sweden: (1) The notion that AMR prevention and AMR‐related human health risks are within the veterinarians’ professional responsibility. (2) A feeling that this is currently agreed on within the profession (in Sweden). (3) A notion that the current AMU is restrictive. (4) Experience of AMU policies as credible and useful. (5) Professional space to effectively treat animals . (6) A notion of precise AMU as an expression of advanced veterinary skill and (7) Good relations with farmers despite restrictive use. It is probably through the combination of these factors that accounting for the AMR‐related public health risk (in an abstract future) and restrictive use of antibiotics can have such a strong position. Thus, just as non‐restrictive or 'misuse' of antibiotics cannot be reduced to a matter of individual behaviour, but is something that needs to be understood in its specific contextAll authors declare they have no conflict of interest.https://www.riksdagen.se/sv/dokument-lagar/dokument/svensk-forfattningssamling/lag-2003460-om-etikprovning-av-forskning-som_sfs-2003-460; accessed 08/11/2021).According to the Swedish Ethical Authority, the study did not require ethical approval because no sensitive personal information was collected (reference ‐ law on ethical review of research involving humans 2003; available at Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.


Vibrio vulnificus and V. parahaemolyticus, found naturally in marine and estuarine environments, are the leading cause of seafood associated gastrointestinal illness and death. Consumption of improperly cooked crabs and handling of live crabs are potential routes of exposure to pathogenic bacteria such as V. vulnificus and V. parahaemolyticus. Little information is available on serotype genetic and antimicrobial profiles of V. vulnificus and V. parahaemolyticus recovered from Maryland estuaries. The aim of the present study was to determine the serotype of V. parahaemolyticus, evaluate antimicrobial susceptibility and genetic profiles of V. vulnificus and V. parahaemolyticus isolated from water and blue crab  samples collected from the Maryland Coastal Bays. One hundred and fifty (150) PCR confirmed V. parahaemolyticus including 52 tdh+ (pathogenic) and 129 V. vulnificus strains were tested for susceptibility to twenty (20) different antibiotics chosen by clinical usage for Vibrio species. The O serogroups were determined using an agglutination test with V. parahaemolyticus antisera. Pulsed-field gel electrophoresis (PFGE) was used for molecular subtyping to investigate the genetic diversity among tested strains. The most prevalent serotypes were O5 (33.3%), O3 (18.7%) and O1 (14.7%). More than 41% of all tested Vibrio isolates were resistant to three or more antibiotics. Cephalothin showed the highest resistance (42% and 61%), followed by cefoxitin (42% and 31%) and ceftazidime (36% and 29%) for V. vulnificus and V. parahaemolyticus, respectively. Most strains (99–100%) were susceptible to ampicillin/sulbactam, levofloxacin, piperacillin, piperacillin/tazobactam, and tetracycline. Fifty percent (50%) of the cephalothin resistant strains were crab isolates. Vibrio vulnificus and V. parahaemolyticus isolates demonstrated a high genetic diversity and 31% of V. vulnificus and 16% of V. parahaemolyticus strains were PFGE untypeable. No correlations were found between the V. parahaemolyticus serotype, pathogenicity, genetic and antimicrobial resistance profiles of both species of Vibrio. The observed high multiple drug resistance of V. vulnificus and V. parahaemolyticus from blue crab and its environment is of public health concern. Therefore, there is a need for frequent antibiotic sensitivity surveillance for Vibrio spp. Vibrio vulnificus and V. parahaemolyticus are Gram negative non-spore forming rod-shaped halophilic bacteria naturally found in warm marine and estuarine environments  and Thermostable Related Hemolysin (TRH) which are determinants of virulent strains (V. vulnificus: a species-specific marker- V. vulnificus hemolysin gene A (vvhA) and virulence correlated gene (vcgC) ( strains . There ae (vcgC) .V. parahaemolyticus and 111 cases of V. vulnificus, 500 hospitalizations and 100 deaths of which 35 are V. vulnificus related deaths are reported every year  were from Maryland  (Pulsed-field gel electrophoresis (PFGE) was used to investigate the genetic variation of the e EDTA)] . The gelV. vulnificus and V. parahaemolyticus isolates, and to evaluate the differences in the occurrence of antimicrobial susceptibility and correlation between the sample sites, types pathogenicity, Genetic and antimicrobial resistance profiles using SPSS version 26.0 .One-Way Analysis of Variance (ANOVA) and Pearson’s Chi square test were used to analyze the data and compare the means among sites, sample type, antibiotic resistance patterns of V. parahaemolyticus strains. Except for O7 and O9, all serogroups were equally distributed in all four sites in Maryland Coastal Bays (tdh+ and 1 tdh+ and trh+). However, there was no correlation between serotype and pathogenicity .Serotype was performed in all 150 tal Bays . The mostal Bays . Twenty-V. vulnificus and 150 V. parahaemolyticus) strains were tested for antibiotic sensitivity. Sixty-five percent (65%) of all tested V. vulnificus and V. parahaemolyticus strains were resistant to one or more classes of antibiotics and 42% were multidrug resistant. The highest resistance was observed for cephalothin, 42% for V. vulnificus and 61% for V. parahaemolyticus, followed by cefoxitin (42 and 31%), ceftazidime (36 and 29%), ceftriaxone (35 and 30%), cefotaxime (27 and 20%), cefepime (21 and 20%), amikacin (20%) and meropenem (16 and 19%) for V. vulnificus and V. parahaemolyticus, respectively. Ninety-five to 100% of both Vibrio species were susceptible to most of the commonly used antibiotics such as ampicillin, ampicillin/sulbactam, doxycycline, levofloxacin, piperacillin, piperacillin/tazobactam, tetracycline and trimethoprim/sulfamethoxazole (p ≤ 0.05) higher cephalothin resistance than those from crab meat and hemolymph  V. vulnificus and 92 (including 28 tdh+) V. parahaemolyticus strains from different sources were selected and a dendrogram was constructed based on SfiI PFGE patterns to compare the banding profiles of these bacteria. Thirty-one percent (31%) of V. vulnificus and 16% of V. parahaemolyticus strains were PFGE untypeable and failed to yield discernible patterns. Both vibrio species demonstrated a high genetic diversity and they were grouped into 8 (A to H) V. vulnificus and 19 (A to R) V. parahaemolyticus clusters of related patterns of ≥ 75% similarity. No relationship was observed between the genetic and antimicrobial resistance profiles of both Vibrio species. Five (5) V. vulnificus  and 10 V. parahaemolyticus  strains showed similar antimicrobial resistance profile, although they were genetically different  antigen using conventional kits. Similar to our results they did not report O7 and O9 serotypes. Besides of O3 group, O4:K12 and O4: K (unknown) are pandemic V. parahaemolyticus strains of the Pacific Northwest associated with outbreaks in New York, Atlantic Coast of Spain in 2012 and in 13 United States Atlantic Coast states in 2013  (p > 0.0%) in the Maryland Coastal Bays. Both Vibrio species showed the highest resistance to cephalothins, one of the recommended antibiotics for vibriosis treatment, followed by cefoxitin, ceftazidime, ceftriaxone, cefotaxime, cefepime, amikacin and meropenem. V. parahaemolyticus strains showed higher resistance to cephalothin compared to V. vulnificus. Resistant to commonly used antibiotic is also elevated in V. parahaemolyticus than V. vulnificus (Vibrio strains (100%) were susceptible to ampicillin/sulbactam, and all V. vulnificus strains were susceptible to levofloxacin, piperacillin, piperacillin/tazobactam, tetracycline and trimethoprim/sulfamethoxazole (p ≤ 0.05) higher cephalothin resistant strains compared to crab meat and hemolymph samples (V. vulnificus and V. parahaemolyticus were found by V. vulnificus (95%) and V. parahaemolyticus (82%) to cephalothin versus 38 and 23% for V. vulnificus and V. parahaemolyticus, respectively observed in the present study.lnificus . All teshoxazole . Half of samples , 3; therV. vulnificus and V. parahaemolyticus strains from crabs and its environments  were performed according to the standard PulseNet PFGE protocol for Vibrio spp. with the restriction enzyme SfiI. Though a few V. vulnificus and V. parahaemolyticus strains recovered from seafood in the United States, specifically in Maryland have been genetically characterized by PFGE analysis, this is the first study that reported the PGFE profiles of V. vulnificus and V. parahaemolyticus strains recovered from crabs and its surrounding environments in the Maryland Coastal Bays.It has been reported that molecular typing is a reliable and useful tool for investigating the genetic diversity and tracking sources of contamination of food and waterborne pathogens in aquatic systems and food processing plants . In thisV. vulnificus were found with different 80% similarity in banding patterns. Multidrug resistant as well as potential pathogenic strains of both species of Vibrio were distributed in different clusters. Only two strains out of 49 V. vulnificus strains fell into the same cluster, with 100% similarity and exhibited the same multidrug resistant pattern. Moreover, these isolates were from the same site (site 9) and source (crab); however, they differed in intermediate antibiotic profile . V. vulnificus showed higher percentage (≥80%) of similarity compare to V. parahaemolyticus (≥75%); however, these differences were not statistically significant (p > 0.05). The PFGE profiles of V. parahaemolyticus (V. parahaemolyticus isolates clustered (A to R) at 75% or higher similarity in banding patterns. These results indicate high intraspecific diversity of this species. Our findings are consistent with a previous study that reported high genetic heterogeneity among V. vulnificus and V. parahaemolyticus isolates recovered from oysters and water during a relaying study in the Chesapeake Bay, Maryland  was higher than V. parahaemolyticus (16%) despite the fact that 50 μM of thiourea was added to 0.5X TBE (Tris-Borate EDTA) buffer for the repeated PFGE experiments. Only 4 V. parahaemolyticus and 1 of V. vulnificus strains were typeable after the addition of thiourea in the electrophoresis buffer. V. vulnificus strain was also reported in studies of PFGE analysis of V. vulnificus strains isolated from Taiwan and the United States  for its treatment. Therefore, frequent antibiotic sensitivity surveillance is needed.The PFGE profiles of The raw data supporting the conclusions of this article will be made available by the authors, without reservation.SP, PC and LS contributed to the conception and design of the study. LS and SO were involved in experimental procedures in the laboratory and data collection and analysis. The manuscript was written by LS, and reviewed, edited, and approved by all authors. SP and PC were responsible for the integrity of the work and overall supervision.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be taken as a potential conflict of interest.


Molecular ballistics combines molecular biological, forensic ballistic, and wound ballistic insights and approaches in the description, collection, objective investigation, and contextualization of the complex patterns of biological evidence that are generated by gunshots at biological targets. Setting out in 2010 with two seminal publications proving the principle that DNA from backspatter collected from inside surfaces of firearms can be retreived and successfully be analyzed, molecular ballistics covered a lot of ground until today. In this review, 10 years later, we begin with a comprehensive description and brief history of the field and lay out its intersections with other forensic disciplines like wound ballistics, forensic molecular biology, blood pattern analysis, and crime scene investigation. In an application guide section, we aim to raise consciousness to backspatter traces and the inside surfaces of firearms as sources of forensic evidence. Covering crime scene practical as well as forensic genetic aspects, we introduce operational requirements and lay out possible procedures, including forensic RNA analysis, when searching for, collecting, analyzing, and contextualizing such trace material. We discuss the intricacies and rationales of ballistic model building, employing different tissue, skin, and bone simulants and the advantages of the “triple-contrast” method in molecular ballistics and give advice on how to stage experimental shootings in molecular ballistic research. Finally, we take a look at future applications and prospects of molecular ballistics. Ballistics, as the science of the motion of projectiles, can be divided into interior ballistics, external ballistics, and terminal ballistics. Interior ballistics studies the projectile while still within the gun; exterior ballistics examines the projectile’s movement through air; terminal ballistics addresses the penetration of and interaction with solids by the missile . TherefoGSR are mainly composed of burnt and unburnt organic or inorganic particles from the explosive primer from the cartridge, the propellant, and possibly fragments of the bullet, cartridge case, and even the firearm, that will frequently contain Sb, Ba, and Pb or Zn, Cu, and Ti . They cawound ballistics which can be considered a subdivision of terminal ballistics concerned with the motions, effects on, and interactions of a projectile with organic tissue [Wound tracks, meanwhile, i.e., the permanent paths that projectiles leave in traversed tissues, as well as entry and exit wounds are the main subjects of c tissue . Wound bc tissue .Molecular ballistics, in contrast, is defined as applying the methods and techniques of forensic molecular biology to the analysis of biological traces that are generated when the interaction of firearm projectiles with hit targets produces “forward spatter” and/or “backspatter.” Such has been termed the biological material that is propelled out of the exit and entrance wound, respectively, of a biological target hit by a firearm projectile. While the emergence out of the exit wound of forward spatter accompanying the projectile is straightforward to comprehend, the formation of backspatter is less intuitive: it is generated due to the combined forces of several interacting wound ballistic effects including (1) the elastic collapse of the temporal wound cavity and the concomitant equilibration of resulting overpressure [pressure –12, (2) pressure , 13, 14,pressure , 15, 16.inner surfaces of firearms for traces of backspatter in routine forensic casework involving gunshot-related crimes [The term “molecular ballistics” was coined when about 10 years ago Courts and Schyma started investigating whether backspatter can be found and recovered from inside surfaces of firearms that had been used to shoot at biological targets. The phenomenon of backspatter had been discovered and described by Weimann as early as 1931 , but CouIn the following years, molecular ballistic research had its scope expanded considerably, and it was demonstrated that not only nuclear DNA but also mitochondrial DNA (mtDNA), messenger-RNA (mRNA), and micro-RNA (miRNA) can simultaneously be recovered and analyzed in parallel from traces of backspatter . Also, iRecently now, Gosch et al. analyzed DNA traces recovered from firearms wielded in realistic, casework-relevant handling scenarios to improve the understanding of factors affecting the variability of trace DNA characteristics recovered from firearms handled in gun-related crimes . By showindividualization and contextualization of traces and trace patterns generated by shots at biological targets which then together with wound ballistic and GSR analyses may critically contribute to an evidence-based, objective reconstruction of the course of events and inference of involved persons.The aim of applying molecular ballistics is to fully and in-depth exploit all available biological evidence from scenes of gun-related crimes and to provide a multi-dimensional molecular analysis to support the The scope of molecular ballistics encompasses all traces of biological material ejected from a body by wound ballistic effects that is hit by a high velocity projectile. In general, the pattern of blood and tissue traces at a scene of gunshot injury is dependent mainly on whence they left the body: from the entry site or, if present, the exit site of the bullet. In technical terms, projectiles that only enter but do not exit the body create “penetrating wounds,” while projectiles that fully traverse and then leave the body or one of its parts create “perforating wounds” , yet theBiological material found at a crime scene involving gunshot injury can be analyzed to help reconstruct contextual details of the course of events of the crime—for example, to infer the number of persons involved or whether the victim was moving or being moved after being hit by the shot. Still, considering each gunshot isolatedly (disregarding complex situations like a projectile perforating an arm and then re-entering the body at another site), there are two directions into which biological material can be propelled, as laid out before: following the projectile through the exit wound in the direction of flight (forward spatter) and from the entry wound in the direction opposite to the trajectory (backspatter). Both exiting and entering projectiles are capable of causing the expulsion and distribution of biological material in comparable angles, yet with different amounts and spatter velocities  causing Depending on the shooting distance, backspatter traces are often found on the shooter’s clothes and hands in particular, whence they can be collected. Yet, as pointed out, the center of molecular ballistics’ interest is the firearm used in gunshot-related crimes and incidents. This central object often connects the victim(s) and the perpetrator, therefore traces recovered from there may be of substantial evidential weight. In fact, backspatter traces can consolidate on basically all outer and inner surfaces of a firearm and when manifested as microspatter (droplet size < 0.5 mm diameter ) may eveEspecially, but not only after contact shots, is it highly advisable to investigate the inside of the barrel for backspatter traces. This inner surface is well protected from environmental contamination and from direct contact with, e.g., packaging material which could lead to loss of biological material . In a syAs forward spatter is trailing the projectile out of the exit wound from perforating shots, unlike backspatter, it cannot consolidate on the shooter or the fired gun. Therefore, forward spatter can only be found on the crime scene (including objects and bystanders) and is a main subject for BPA in investigation of gunshot-related crimes. Consequently, documenting and securing traces of forward spatter is a task for crime scene investigation personnel who should be (made) aware of the significance of these traces. While no molecular ballistic research focusing solely on forward spatter has been published so far, all information gathered from the molecular biological analysis of such traces can be compared and complemented with information from traces collected from absent and later secured guns, perpetrators, or perhaps even victims. Also, in cases where the circumstances of a crime are unclear or evidence or witness reports are contradictory, molecular ballistics employing forensic RNA analysis  can support contextualized event reconstruction by the molecular differentiation between tissue origins in traces of forward spatter where BPA has been the only option so far, e.g., to help the discrimination between blunt impact or gunshots .There are two main questions of criminalistic relevance connecting shooting distance and backspatter. (1) Up to what distance can traces of backspatter be recovered from the firearm and/or the shooter and what variables  may influence this? (2) Is there a correlation between shooting distance and (any aspect of) backspatter that can be used to infer the former from the latter?To answer these questions, several studies have been conducted so far. For instance, Karger et al. performed experiments using a 9-mm SIG P210 pistol and two kinds of 9-mm Luger ammunition to shoot at the heads of calves from distances of 0 to 10 cm and found instances of macrobackspatter (droplet size > 0.5 mm diameter) and microbackspatter (droplet size < 0.5 mm diameter) up to distances of 119 cm and 69 cm, respectively , 43. In As firearms are—with very few exceptions—made of metal or alloys with antirust or anticorrosive coatings like browning gas nitriding, teniferation (ferritic nitrocarburizing), etc., all sampling techniques are applicable that are recommended for non-porous/non-absorbent materials. To this date, a comprehensive study to establish the optimal sampling technique, comparing swab brands and material as well as extraction methods for different biological materials  potentially comprised in backspatter traces has not been performed. In a rather superficial study, Wood et al. still found that the recovery from acellular human DNA with both cotton and nylon swabs from “firearm metal” (sic) was inefficient, only at about 15% of the applied amount of DNA . Therefoalways be performed in parallel to sample collection for standard forensic DNA analysis, e.g., from the firearm’s grip, trigger, etc. to facilitate individualization which is essential for inferring the weapon’s handler. How “touch DNA”  is transferred to a gun by its handler(s) via touch/direct contact in different realistic mock case scenarios and how to collect it has recently been investigated in-depth by Gosch et al. [At this point, it needs to be emphasized that the recovery of backspatter traces for molecular ballistic analyses should h et al.  as mentiThe search for and collection of traces should be performed “from the outside to the inside,” i.e., first, all outer surfaced should be carefully investigated and sampled, even where no traces are visible (minding “touch DNA” and microspatter). Afterwards, the weapon should be disassembled (if possible) and samples should be retrieved from detachable weapon parts, especially from “inner” surfaces , which have proven to bear DNA-containing material that allows for successful victim identification . In pistThe inside of the barrel, on the other hand, is the surface not only best protected from the outside but also the most difficult to sample, as pressure required for efficient sample collection can hardly be applied onto the swab. Additional methods should thus be considered in cases where traces are detected  but cannot properly be recovered using swabs which will frequently be the case with long barreled firearms. For instance, a patented tool devised and specially dedicated for the efficient and quantitative collection of backspatter traces from inside gun barrels is called “GunSwab C1” which is available for handguns and long barreled guns of different calibers . The device consists of DNA-free felt pieces attached to a metal pulling rope that can be pulled through the entire length of the barrel, thereby applying pressure and brushing loose and collecting traces and residues sticking to the barrel surface.In simple terms, in molecular ballistics, DNA-based individualization of biological material should help to answer the question “Who shot whom?” and short tandem repeat (STR) profiling from backspatter traces has already been shown to enable identification of victims of gun violence, even from complex mixed traces . DNA proDifferent methods have been employed to extract DNA from backspatter samples collected from firearms, but given that neither has a comparative study of different methods as yet been performed, nor is exact replication of the backspatter generation even in replicate shots under identical conditions possible , it cannThe origin of forensic RNA analysis may be traced back to 1994 when Phang et al. were first to use RT-PCR to analyze mRNA from postmortem tissues in a forensic setting . Since tCurrently, however, and in contrast to DNA analysis, there is no validated commercially available kit for forensic RNA extraction, and as shown by Grabmüller et al.  and SchwIn general, if RNA analysis is to be performed, samples that cannot immediately be processed after collection should be immersed in an RNA-stabilizing reagent  and/or stored at −80°C. If such facilities are unavailable, samples should be stored at no more than room temperature, kept dry and in the dark, and be processed as soon as possible. RNA and DNA can then be co-extracted from the same sample without having to choose DNA over RNA or vice versa , 63. If always when experimental shootings at (molecular) ballistic models are performed, backspattered material and/or splinters or fragments from the bone (simulant)s or even slowed down bullets ricocheting back from the bullet trap may potentially hit and injure the shooter. We strongly advise to adhere to all applicable safety regulations and always have the shooter wear appropriate protective gear all the time.Be aware, that similarity in the deceleration of the projectile between the simulant and the living tissue the simulant has been validated forsimilarity in the deformation behavior of the projectilesimilarity in the kinetic energy dissipationkinetic energy dissipation measurability with reasonable accuracyextrapolation of temporary cavity diameterelastic behavior similar to living tissue for observation and measurement of temporary cavity formation and tissue compressionextrapolation of permanent cavity diameterreproducibility.The often-cited phrase attributed to the British statistician George Box, “All models are wrong, but some are useful” is extraordinarily fitting in the context of establishing models for molecular ballistic research or ballistic research involving biological targets in general. The requirements for a realistic model in this field are quite high, as the human body is a complex, heterogeneous structure with considerable inter-individual variation. Persons having their body donated to ballistic research after their death are exceedingly rare. Other biological targets like (dead) animals, animal parts, or animal tissues have been employed in studies but are difficult to replicate due to biological variation and will at best only be a mediocre simulation of the human body and its parts. As an alternative, artificial materials as simulants for biological tissues and structures are available and still being developed that exhibit physical and mechanical properties comparable to the biologic original, and while a perfect simulation of (parts of) the human body in all its complexity and detail including the vascular system appears to be out of reach, those simulants can still be used to compose a model suitable for ballistic tests investigating specific aspects. Already in 2004, Jusilla compiled a list of qualities needed for tissue simulants , which isimilarity in the generation of forward- and backspatter a source of biological material (tissue/blood), which must in some way be integrated into the model system.All those requirements should be fulfilled by the models employed in molecular ballistics research; however, further aspects must necessarily be included:To instantiate all qualities in a single model setup requires an extensive developmental effort, time, costs, and thorough validation with, if possible, several types of firearms. Research facilities with less resources may instead choose to use more simplistic models, implicating only the most important aspects which are needed to simulate the part of the body the planned ballistic test is aiming for. Essentially, these are the primarily involved tissue (muscle or organ), the skeletal part if head, chest, or limbs are considered (represented as a bone structure), and the surrounding skin.Earlier, less sophisticated models used for molecular ballistic experiments usually consisted of mere gelatin blocks, sponges and acrylic spheres , or gelaOther model systems for ballistic experimentation could be adapted for use in molecular ballistics by adding a source of biological material: For example, Riva et al. presented individual synthetic head models based on real cases of gunshots to the head by using polyurethane plates, ballistic soap, and gelatin with proportions reflecting the respective victims’ tissues as measured by postmortem CCT . Thali eAn overview of simulant materials that are or were used or investigated in the context of (molecular) ballistics is given in Table All studies on molecular ballistics that are currently available used 10% ballistic gelatin as tissue simulant , 51–53. Except for torso shot simulations, where bone structures need not be included, and for shots merely focusing on the analysis of, e.g., temporal cavity characterization or gelatin behavior, a solid material bone simulant should be implemented in ballistic model setup. Especially when investigating shots to the head as the most important target area in (molecular) ballistic research, a rigidly cased model is needed, as full casing will strongly influence energy distribution and temporal cavity formation and consequently forward and backspatter behavior , 87. EarFor molecular ballistic studies, skin simulants have not been thoroughly evaluated and compared so far. Still, especially when investigating contact shots and/or head shot simulations, the skin/scalp serves an important role for backspatter generation by enabling the simulation of a subcutaneous gas pocket . When deJusilla et al. also relied on these values and tested different materials, concluding that cowhide with semi-finished chrome-tanned upholstery “crust” most closely emulates human skin, while acknowledging the general problem with variation in biological simulants . The skiTo aid investigations and sample collection after gunshot incidents, it is essential to know about the mechanisms influencing forward or backspatter generation and where the traces are to be found. After successfully having tested paint  and radiological contrast material in ballistic models –101, SchAn important aspect in molecular ballistic experimentation is an effective cleaning procedure to remove all remaining and especially biological traces before performing a subsequent shot with the same gun. Also, cleaning effectiveness should always be assessed by taking negative samples from the freshly cleaned gun. Methods and materials employed in the cleaning procedure should be suitable to be applied at the shooting site thus are neither excessively time consuming nor too complicated. Care should be taken to avoid that the procedure damages or destroys the gun’s alloy, coating, or other parts. This should be checked in advance, and we advise to obtain information about the materials and manufacturing process of the weapons to be used and to test whether, for instance, a cleaning agent intended to remove traces of nucleic acids may fret at the gun’s surfaces. Consequently, studies on molecular ballistics describe mechanical cleaning as the method of choice, employing pieces of woolen felt for cleaning the inside of the barrel, and different chemical substances, like Roti® Nucleic Acid-Free  , 38, DNA“Cold cases” are unsolved criminal investigations which remain open pending the discovery of new evidence. It seems promising to apply molecular ballistics to the revision of such cases with gun-related injuries or deaths if the firearm in question had not been probed from the inside during the original investigation, which will regularly be the case. Backspatter on inner surfaces may then still persist, can be recovered and analyzed after years of storage, and can remain analyzable even after standard ballistic test shootings have been performed with the gun . For insMassive parallel sequencing (MPS), also often referred to as “next-generation sequencing” (NGS), is an emerging key technology in genetics and genomics  enablingBlank guns, or blank firing/cartridge guns, are underrepresented as a research subject and underrated as a threat to human beings. There are numerous reports on cases and incidents of injury and death involving these weapons suggesting a substantial hazard potential, which are presented comprehensively in a recent study on the analysis of backspatter generated by blank gun contact shots at ballistic models by Euteneuer et al. . They de